Instructions for use

Title Comprehensive studies of organic synthesis by utilizing chemical features of natural products

Author(s) ZETRYANA PUTERI TACHRIM

Citation 北海道大学. 博士(農学) 甲第13596号

Issue Date 2019-03-25

DOI 10.14943/doctoral.k13596

Doc URL http://hdl.handle.net/2115/74144

Type theses (doctoral)

File Information Zetryana_Puteri_Tachrim.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

Comprehensive studies of organic synthesis by

utilizing chemical features of natural products

(天然物の化学的特性を活かした有機合成反応の網羅的検討)

Doctoral dissertation

(The Special Postgraduate Program in Biosphere Sustainability Science)

Zetryana Puteri Tachrim

ゼトリヤナプテリターリム

Hokkaido University, Graduate School of Agriculture

Division of Applied Bioscience, Doctor Course

Japan

2019.03

i

Acknowledgements

Bismillahirrahmannirrahhim

I would like to send my first regards to Allah SWT, the Lord of this universe, that gave

a lot of bless to me, therefore I could finish my master course study. Then, the beloved

prophet Muhammad saw.—the truth role model to lead human kind for better world.

My best honor is send to Dr. Makoto Hashimoto who guide and supervised me from the

beginning of my master course until my graduation. He gave me a lot of chance to explore my

experience in research, such as journal construction and submission; oral presentation at

International conference at Sanya, China 2016 and Osaka, Japan 2018; JSBBA Hokkaido

branch student and poster award 2017 during my Doctoral course. Moreover, I would like to

thank Prof. Hashidoko and Dr. Sakihama for their advices during my study in the Ecological

and Molecular Chemistry Laboratory. I would like to send my greatest thanks for all

supervisors, especially Prof. Matsuura, who giving me the advice for my Doctoral dissertation.

I would like to say my greatest thank you to LPDP (Indonesia Endowment Fund for

Education) for all the financial support: university fee, living allowance, conference fee, and

experimental fee, etc. Because of LPDP, I am proud of being Indonesian and can obtain the

best opportunity in my life.

I would like to send great honor to Prof. Endang Asijati W. and Dr. Meri S. that give me

great support from the beginning of my study. My regards to Wang Lei for supports my

experiments. Zaki and Mami as my student supporter that helped me to adjust all inquires

when my first arrival at Sapporo. Many thank you for Ecochemistry lab members: Wang,

Yoshida, Oida, Hashi, Kuro, Haru, Riri that support my study. I would like to send my

gratitude for all the support from all my International friends, Indonesian friends, LPDP

awardee, all LPDP organizers, and all my previous bachelor degree friends.

My family: Edya Tachrim, Yohana Sian, Putri Syarita, Yodytian Putera, Detri Linda

Sian, and all my family that I cannot mention them. Thank you for your support and pray for

me. The last, to all people that kindly to help me and all names that I can not mentioned it one

by one.

ii

And if whatever trees upon the earth were pens and the sea [was ink], replenished

thereafter by seven [more] seas, the words of Allah would not be exhausted. Indeed, Allah is

Exalted in Might and Wise (Al-Qur’an, 31: 27). The greatest things only own by God and all

the knowledge or research that we found actually already His created.

January 9th

, 2019

Zetryana Puteri Tachrim

This Doctoral dissertation is dedicated to:

1. My father who prohibited me to go abroad at the beginning and always accompany me

during scholarship interview.

2. My mother who always forced me to come to the lab early or during weekend, but

always believe that I can finish everything in a good way which bless by God.

In memorial to my best friend Ika Novianingsih that I last met her in February 2016

“You just need to study at school; therefore, you will not get discouraged in front of great

people”—My Grandfather H. Muh. Sian

iii

Abbreviation

1H-NMR Proton Nuclear Magnetic Resonance

13C-NMR

Carbon Nuclear Magnetic Resonance

1D-NMR One-Dimensional Nuclear Magnetic

Resonance

2D-NMR Two-Dimensional Nuclear Magnetic

Resonance

COSY Correlated Spectroscopy

HMQC Heteronuclear Multiple-Quantum

Correlation Spectroscopy

HMBC Heteronuclear Multiple Bond

Correlation Spectroscopy

HETCOR Heteronuclear Correlation Spectroscopy

NOESY Nuclear Overhauser Effect

Spectroscopy

TOCSY Total Correlation Spectroscopy

TFA-d Deuterated trifluoroacetic acid

TfOH Triflic acid

TfOD Deuterated triflic acid

H/D Hydrogen/deuterium

TFA- Trifluoroacetyl-

OSu N-hydroxysuccinimide ester

WSCD-HCl Water-Soluble Carbodiimide

Hydrochloride

NHS N-hydroxysuccinimide

Ar Arene

L-/D-Ile L-/D-Isoleucine

L-/D-allo-Ile L-/D-allo-Isoleusine

Gly Glycine

L-/D-Ala L-/D-Alanine

L-/D-Val L-/D-Valine

L-/D-Leu L-/D-Leucine

L-/D-Nva L-/D-Norvaline

L-/D-Nle L-/D-Norleucine

L-/D-Pro L-/D-Proline

L-/D-Phe L-/D-Phenylalanine

L-/D-Tyr L-/D-Tyrosine

Pyr N-pyrrole

N-Me Pyr N-methyl pyrrole

DCl Deuterium chloride

1

Table of contents

Acknowledgements……………………………………………………………………………i

Abbreviation………………………………………………………………………………….iii

Table of content……………………………………………………………………………….1

Chapter 1 Introduction and outline of the study .................................................................3

1.1 Introduction ............................................................................................................................ 3

1.1.1 Direct halogenations of carbohydrate’s primary alcohols ............................................ 3

1.1.2 α-Amino acids chiral center and stereochemistry: unique consideration

for direct acylation to synthesize -amino aryl ketone via Friedel–Crafts

acylation .................................................................................................................... 7

1.1.3 Hydrogen/deuterium exchange: Introducing deuterium isotope into

aromatic moiety by acid catalyst .............................................................................. 10

1.2 Outline of the study .............................................................................................................. 12

1.2.1 Direct halogenation at primary alcohols of carbohydrate .......................................... 12

1.2.2 α-Amino acids extensive acylation ........................................................................... 13

1.2.3 Hydrogen/deuterium exchange of cycloDOPA derivatives ....................................... 15

Chapter 2 Direct halogenations at primary alcohols of carbohydrate.............................. 16

2.1 Introduction .......................................................................................................................... 16

2.2 Results and Discussion ......................................................................................................... 18

2.2.1 Direct halogenations at primary alcohols of sucrose ................................................. 19

2.2.2 Direct halogenations at primary alcohols of 1-kestose .............................................. 37

2.3 Experimental section ............................................................................................................ 44

2.3.1 Sucrose re-subjection into Appel reaction ................................................................. 44

2.3.2 Chemoenzymatic Synthesis of 1′-Halodeoxysucrose Derivatives .............................. 47

2.3.3 1-Kestose subjection into Appel Reaction ................................................................ 48

2.3.4 Acetylation of Sucralose .......................................................................................... 49

2.3.5 Acetylation of 1-kestose ........................................................................................... 50

2.3.6 Deacetylation of per-O-acetylated halogenated carbohydrates .................................. 50

2.3.7 1H

and

13C NMR Literature Comparison of Halodeoxysucrose

Derivatives with Observation Data ........................................................................... 55

2.3.8 1H

and

13C NMR Literature Comparison of Halodeoxy-1-kestose

Derivatives with Observation Data ........................................................................... 77

2.4 Conclusion ........................................................................................................................... 83

Chapter 3 -Amino acid extensive acylation ..................................................................... 84

3.1 Introduction .......................................................................................................................... 84

3.2 Result and discussion ........................................................................................................... 86

2

3.2.1 Friedel–Crafts Acylation of aliphatic -amino acid derivatives:

Isoleucine derivatives synthesis and modification ..................................................... 86

3.2.2 Friedel–Crafts Acylation of aliphatic -amino acid derivatives: Synthesis

and modification of various aliphatic -amino acid derivatives ................................ 96

3.2.3 Friedel–Crafts acylation of aromatic α-amino acid derivatives: Synthesis

and modification of phenylalanine and tyrosine derivatives synthesis and

modification........................................................................................................... 107

3.3 Experimental section .......................................................................................................... 118

3.3.1 General procedure for the preparation of TFA--amino acid .................................. 118

3.3.2 Procedure for the preparation of TFA-L-/D-tyrosine (L-/D-98) ............................... 123

3.3.3 General Procedure for the Preparation of TFA--Amino Acid N-

Hydroxysuccinimide Ester ..................................................................................... 123

3.3.4 General procedure for the preparation of TFA-protected -amino aryl-

ketones .................................................................................................................. 129

3.4 Conclusion ......................................................................................................................... 141

Chapter 4 Hydrogen/deuterium exchange of aromatic compounds (cycloDOPA

derivatives) ........................................................................................................................ 142

4.1 Introduction ........................................................................................................................ 142

4.2 Results and Discussion ....................................................................................................... 143

4.3 Experimental section .......................................................................................................... 152

4.4 Conclusion ......................................................................................................................... 154

Chapter 5 Conclusion and future prospect ...................................................................... 155

Reference ........................................................................................................................... 157

3

Chapter 1 Introduction and outline of the study

1.1 Introduction

Natural products hold a unique and enormous chemical diversity of which individual

scaffolds have different reactivity and stability, chemical modification potentials, or specific

functional groups at special position that involves in molecular interaction. The existence of

chiral centers and stereochemistry are also important key in biomolecules that critically

influence the properties of natural products. Organic synthesis gives opportunity for exploring

a challenging molecular architecture that extent the discovery and invention of new synthetic

strategies, methods, and analyses. Taking advantage from vast chemical features of

natural products in organic synthesis, a wider range of applications and a comprehensive

study for multipurpose in research and development are possibly achieved.

1.1.1 Direct halogenations of carbohydrate’s primary alcohols

Carbohydrate is the most abundant organic compounds in nature. In plant, carbohydrates

act as chemical energy (glucose, starch, glycogen); are components of supportive structures in

plants (cellulose), crustacean shells (chitin), and connective tissues in animals

(glucosaminoglycans); and are essential components of nucleic acids (D-ribose and 2-deoxy-

D-ribose). The chemistry of carbohydrates is essentially the chemistry of hydroxy groups and

carbonyl groups and of the acetal bonds formed between these two functional groups.1

In midst of carbohydrates, sucrose (Figure 1) is produced worldwide in large quantities.

This compound is highly functionalized molecules with complex stereochemistry, due to the

presence of several hydroxylated stereogenic centers.2 Sucrose is also accepted as a standard

of sweet taste quality. Hence, the sweetness intensity and other taste properties of natural and

synthetic sweeteners are frequently compared with sucrose. Interestingly, substitutions of

halogen into certain carbohydrate skeleton gave different relative sweetness than sucrose

(Table 1).3 For example, 4,1′,6′-trichloro-4,l′,6′-trideoxygalactosucrose (Table 1)—known as

sucralose—is daily used as one of the alternative sweetener that can enhance 2000-fold

sweetness activity than sucrose. Moreover, selective halogenation on other primary center of

sucrose offered the control of sweetness (Table 1). This will lead to the needs for selective

synthesis of sucrose derivatives laborious with simple procedures.

4

Figure 1 Example of selected carbohydrates structure

Table 1 Relative sweetness of sucrose and galactosucrose derivatives3

Compound Sweetness

1′-chloro-1′-deoxysucrose 20

6-chloro-6-deoxysucrose Bitter

6′-chloro-6′-deoxysucrose 20

1′,6′-dichloro-1′,6′-dideoxysucrose 500

6,6′-dichloro-6,6′-dideoxysucrose Not sweet

1′,4,6′-trichloro-1′,4,6′-trideoxygalactosucrose 2000

Sucrose 1

Galactosucrose Not sweet

The syntheses of halogenated sucrose at its primary positions, particularly those

containing bromine and chlorine have been already studied well. Several approaches had been

used for halo substituted from direct or protected hydroxy group of sucrose. Indirect methods

such as the intermediate utilization of 6,6′-ditosyl intermediate;4 selective cleavage of fully

protected sucrose before further halogenation of the free primary alcohol counterpart;5–7

or

bulky functional groups protection and unprotection of one or two primary hydroxy groups8–10

are reported. Then the direct replacement of primary hydroxy groups with halogen, e.g. direct

chlorination of sucrose by well-known sulphuryl chloride (Table 2 : Method A)11

that by the

strict control of reaction condition at –78 oC, the halogenations position will give high

disaccharide

oligosaccharide

polysaccharide

5

selectivity for monochlorinated at 6′-position. In contrast, at room temperature or higher

temperature condition, sulphuryl chloride is not selective due to the direct 4′-chlorination12

of

sucrose observed.

Table 2 Variance of methods for direct replacement only at sucrose primary hydroxy groups

by a halogen substituent

Method Reference Reagents Product (%Yield)

Aa Ref.

11

SO2Cl2 (21.2 equiv.),Py,

CHCl3

6,6′-dichloro-6,6′-dideoxysucrose

hexaacetate (29%);

6′-chloro-6′-deoxysucrose

heptaacetate (43%)

B Ref.13

CH3SO2Br (17.2 equiv.),

DMF

6,6′-dibromo-6,6′-dideoxysucrose

hexaacetate (24%)

C Ref.13

NBrS (5 equiv.), PPh3 (4.5

equiv.), DMF

6,6′-dibromo-6,6′-dideoxysucrose

hexaacetate (21%)

D Ref.13

CH3SO2Cl (17.2 equiv.),

DMF

6,6′-dichloro-6,6′-dideoxysucrose

hexaacetate (51%)

E Ref.13

NCS (6.2 equiv.), PPh3 (6.2

equiv.), DMF

6,6′-dichloro-6,6′-dideoxysucrose

hexaacetate (69%)

F Ref.14

1. (Me2N)3P (27 equiv.),

CCl4 (6.6 equiv.), DMFb

2. KPF6(aq), Et4NBr

(3 equiv.), DMF

6,6′-dibromo-6,6′-dideoxysucrose

hexaacetate (12%);

6-bromo-6-deoxysucrose

heptaacetate (34%)

G Ref.14

1. (Me2N)3P (27 equiv.),

CCl4 (6.6 equiv.), DMFb

2. KPF6(aq), Me4NCl

(6 equiv.), DMF

6,6′-dichloro-6,6′-dideoxysucrose

hexaacetate (15%);

6-chloro-6-deoxysucrose

heptaacetate (45.5%)

H Ref.15

CCl4 (3 equiv.),

PPh3 (6 equiv.), Py

6,6′-dichloro-6,6′-dideoxysucrose

hexaacetate (92%) a

Reaction mixture was maintained at –78 oC, poured into ice-cold sulphuric acid before

purification, and conventional acetylation was conducted after column chromatography.

Reactions at room temperature and 50 oC were resulted in 4,6,1′,6′-tetrachloro sucrose and

4,6,1′,4′,6′-pentachloro sucrose, respectively, in their 2,3-sulphate form. b Conversion of

RCH2OP+(NMe2)3Cl

- before activation by KPF6(aq).

The resulted RCH2OP

+(NMe2)3PF3

- then

treated with the anion (Cl- or Br

-).

Direct halogenation without any protection on hydroxy group of sucrose is a challenging

method for further study. Not only sulphuryl chloride, several other reagents13,14

are available

for the direct and selective replacement at primary hydroxy groups of sucrose by a halogen

6

substituent (Table 2: Method B–G). Unfortunately, these reagents might give a low yield in

many experiments suggesting that acetal migration can occur and reagent purification steps

may involve further chemical modification.15

The high selectivity method within a mild

reaction condition is needed for efficient method of carbohydrate-based products. However,

the use of triphenylphosphine and carbon tetrachloride has been shown to be an efficient

chlorinating reagent for sucrose (Table 2: Method H). The high selectivity for primary

hydroxy group has been associated with a bulky halogenating complex formed from

triphenylphosphine dihalide (Ph3PCX2) and pyridine.16

This method is known as Appel

reaction which is expected as an efficient method for establishment of halogenated sucrose at

the primary positions will be useful to expend the novelty and diversity of carbohydrate-based

products.

Selective halogenation by limiting amount of Appel reagents is particularly complicated,

but separation of monoproduct from the mixture became another issue. Although many

studies have successfully halogenated sucrose at the primary positions4,5,7,9,10,14,17–19

during

past decades, the properties of all former halodeoxysucrose derivatives were limited,

especially for the NMR analyses. NMR analyses are important to describe the substitution site,

but many reports are mainly focused the halogenation selectivity by consideration on identical

comparison of optical rotation with the previous literature data which made the

regioselectivity for sucrose as obscure due to unclear structural identifications of each

monohalogenated sucrose (especially its halomethylene portions). Therefore, the needs to

comprehensive analysis of all monohalogenated sucrose are essential. The objective focused

in this study is to re-subject sucrose into Appel reaction, by following previous condition9 to

afford the mono- and di-substituted products at 6- and/or 6′-positions. The 1′-

monohalogenated sucrose was exclusively synthesized via chemoenzymatic reaction. Thus,

allowing the comprehensive study of structure elucidation by extensive NMR analyses of

halogenated sucroses at the primary positions, the regioselectivity of Appel reaction on

sucrose primary alcohols was investigated.

Since the disaccharide, such as sucrose, already shown its potencies for direct

halogenations from unprotected sucrose, many of researchers also challenged the interesting

properties of longer chain of carbohydrate. Hough and co-worker20

reported the subjection of

raffinose (Figure 1) into sulphuryl chloride. Unlike previous utilization of Appel reagent in

sucrose that selective only for primary alcohols, chlorinated proportion at the primary

positions followed by chlorination at the 4-position of the galactopyranosyl moiety secondary

alcohols was found in this typical reaction. Application of Appel reaction to melezitose for

7

direct chlorination only at primary alcohols is reported.21

This result indicated that Appel

reaction is superior for selective halogenations of carbohydrate primary alcohols.

Short chain fructooligosaccharide (FOS) of inulin type (Figure 1) is categorized as non-

digestible prebiotic oligosaccharide.22,23

In midst of FOS, 1-kestose is reported to have more

sweetening power24

and this great property can be explore further if its primary alcohol is

modified with halogen. The 1′-kestose that elaborated with additional fructose moiety at 1′-

position from sucrose is then tried for halogenations through Appel reaction. Here in this

study, the subjection of 1-kestose to Appel reagent is expected to introduce direct

halogenations into its primary hydroxy groups which the full structural elucidation by

utilizing 1D- and 2D-NMR can determine the precise halogenated position and can contribute

to the synthesis of novel halogenated 1-kestose derivatives.

1.1.2 -Amino acids chiral center and stereochemistry: unique consideration for direct

acylation to synthesize -amino aryl ketone via Friedel–Crafts acylation

The -amino acids are the most significant amino acids in the biological world, because

they are the monomers from which proteins are constructed. Except for glycine, all protein-

derived amino acids have at least one chiral center and the vast majority of

-amino acids in ecosystem are of the L-series.1 Enantiomerically pure N-protected α-amino-

aryl ketones are important building blocks to construct various biologically active

compounds.25,26

The most popular method for its synthesis, Weinreb amide,27

is considerably

less efficient due to the limitations of Grignard or organolithium reagents that apparently are

non-commercially available and under a strong base condition that might hamper synthesis of

the base sensitive N-protecting group of α-amino-aryl ketones. The Friedel–Crafts

reaction25,28–32

becomes a favorable method for synthesis of N-protected α-amino-aryl

ketones33,34

since direct acylation can be undergone with convenient optically pure α-amino

acid derivatives as skeletons into various substrates with a satisfactory yield of product

(Figure 2).

Figure 2 N-protected α-amino-aryl ketone synthesis via Friedel–Crafts acylation by

utilization of optical active N-protected α-amino acids. “PG” stands for protecting group; “L”

stands for as leaving group.

8

In the field of natural products chemical modification, ascertaining which intermediate

has formed is a crucial step in preventing the formation of a racemic adduct during the

reaction. In conventional Friedel–Crafts acylation, aromatic derivatives are reacted with acyl

donor that is activated by BrØnsted or Lewis acid as catalyst.29,35

If -amino acids are

modified and utilized as acyl donor for this typical reaction, the reaction pathway can produce

several molecular species and several considerations should to be occured. Selection of

suitable conditions for these reactions is necessary, especially the reactions for optically active

amino acids. Several plausible routes using various active species36–38

for aromatic acylation

exist (Figure 3). First, if the -proton is deprotonated, and then ketene proportion may be

formed, leading to the loss of chiral retention (Figure 3 (a)). Next, if no proton is eliminated

during the process, the dissociated acylium cation seems more reliably to be formed (Figure 3

(b)). Thus, ascertaining what intermediate has been formed is a crucial step in preventing the

formation of a racemic adduct during acylation.

Figure 3 Plausible acylation routes of aromatic derivatives with modified -amino acids with

leaving group via (a) ketene or (b) acylium cation formation. “L” stands for as leaving

group.34

The identification of chirality for α-amino acids is normally conducted with their optical

rotations and chiral high-performance liquid chromatography (HPLC). But the optical

rotations are sometimes influenced by the measurement condition. It is also very difficult to

set up condition of chiral HPLC for unreported compounds. Therefore, the demonstration of

optically active isoleucine, which has two chiral centers in the molecules, and its diastereomer

allo-isoleucine to identify chilarity of the Friedel–Crafts acylation product by 1H-NMR, might

offer more efficient method. The application of α-amino acid modified with potential leaving

group for synthesis of α-amino aryl-ketone is assumed to be retarded its chirality which might

act as useful acyl donor for Friedel–Crafts acylation.

9

Figure 4 Structures of isoleucine and its diastereomer, allo-isoleucine

In nature, L-isoleucine is usually found in living organism, meanwhile its diastereomer,

D-allo-isoleucine, is commonly found in certain antibiotic and microbial peptide.39

L-

Isoleucine is known to be able to undergo racemization at α-position to produce D-allo-

isoleucine. In geochronology, the rate of racemization reaction of L-isoleucine into D-allo-

isoleucine is relatively slow, which suitable for the analysis.40

As for chiral N-protected α-

amino aryl-ketone synthesis, especially the reaction that involving direct acylation of α-amino

acids, the chirality of product is commonly determined by complexation with chiral shift

reagent25

which racemic α-amino aryl-ketone can be detected by appearance of two separated

proton signals by nuclear magnetic resonance (NMR).

Up to now, there is no reported study that has been checked the chirality of the α-amino

aryl-ketone based on isoleucine and its diastereomer allo-isoleucine. Since the asymmetric

carbon at α-position within these stereoisomers can be observed by 1H-NMR,

41 utilization

within isoleucine and its diastereomer allo-isoleucine (four types of stereoisomers, Figure 4)

might be useful for describe a change in configuration of their chiral center during the

chemical modification. Consideration of chiral center in -amino acids and stereochemistry

for direct acylation to synthesis amino aryl ketone by utilizing isoleucine and its diastereomer

allo-isoleucine can reveal the exploration of activated C-terminal of N-protected amino acid

as acyl donor in Friedel–Crafts acylation using conventional catalyst of AlCl3 (Figure 2).

These chemical features of isoleucine and its diastereomer allo-isoleucine lead to the the new

approach for synthesis of -amino aryl ketone. Utilization of -amino N-hydroxysuccinimide

L-Isoleucine (L-Ile)

Enantiomer

Diastereomer

Epimer at α-position

D-Isoleucine (D-Ile)

L-allo-Isoleucine

(L-allo-Ile)

D-allo-Isoleucine (D-

allo-Ile)

10

ester as a representative acyl donor in Friedel–Crafts acylation is introduced which will be

described later in this thesis.

1.1.3 Hydrogen/deuterium exchange: Introducing deuterium isotope into aromatic moiety

by acid catalyst

H/D exchange is known as one of the method for investigations of reaction mechanisms

to clear ketene formation (or acylium cation formation, Figure 3). It also can be used for

selective protonation/deuteration, which can greatly simplify the spectra of even the largest

protein moieties.42

Hydrogen/deuterium (H/D) exchange methods can generally be divided

into three categories: 1) utilization of D2O as the deuterium source; 2) the involvement of the

addition of an acid or base; 3) the use of metal-promoted H/D exchange. Commonly,

heterogeneous catalytic systems are frequently used for H/D exchange which conducts under

harsh conditions and followed by dehalogenation, hydrogenation, hydrolysis, as well as

epimerization and racemization.43,44

A homogenous system is prefered in H/D exchange,

especially in the case of optically active amino acids, the organic solvent must be considered

for not causing any amino acid racemization during the reaction.

In midst of acid utilization for H/D exchange in aromatic moiety of natural products, a

study that used a D2O/D2SO4 system found that conditions will lead to either partial or

complete racemization of phenylalanine H/D exchange, which is indicated by the exchange of

α-protons.45

Deuterated trifluoroacetic acid (TFA-d) is appropriate for slow H/D exchange,46

but its application to aromatic amides and amines47

is less effective for more electron-poor

substrates such as acetophenone and its derivatives.48

Triflic acid (TfOH) is known as a

catalyst for direct C-acylation and almost all solvent for -amino acids. The utilization of its

deuterated counterpart, deuterated triflic acid (TfOD), as a source of deuterium in H/D

exchange is known for conducting mild reaction condition for aromatic substrates which

advance to be used for several -amino acids without loss of chirality.34

Scheme 1 TfOD-catalyzed C-acylation of toluene and L-glutamic acid derivatives49

11

An example of TfOD as catalysis, solvent, and also deuterium souce of the direct C-

acylation of toluene and glutamic acid derivatives is shown in Scheme 1.49

Based on this

reaction, activation by TfOD can result in typical acylium cation that can prevent any loss of

chirality of optically active starting material (Figure 3 (b)). A fine yield of the acylated

product (94%) from the electrophilic aromatic substitution represents in this reaction which

the rapid H/D exchange and high deuterium incorporation of aromatic moiety can be detected

(68%–75% deuterium incorporation). In Scheme 2, a comparison also can be made between

L-phenylalanine and L-tyrosine treated with TFA-d48

and TfOD.49,50

It shows that the aromatic

proton deuterium incorporation is generally larger than treatment with TFA-d when TfOD is

used. Thus, the absence of α-proton exchange with TfOD indicates no loss of optical purity of

amino acids and found to be superior for mild H/D exchange of aromatic compounds.

Scheme 2 H/D exchange in L-phenylalanine and L-tyrosine. Numbers in brackets give the

percentage of deuterium in the side chain sites.48–50

These advantages are considered potential for broaden the application and structure

elucidation of natural products, such as cycloDOPA (5,6-dihydroxyindoline-2-carboxylic

acid, cDOPA, leukodopachrome). cDOPA is one of metabolites derived from tyrosine, one of

intermediate in melanin formation (mammalian), and betanidin main skeleton (betalain

pigment in plant). The exploration of H/D exchange of cDOPA by utilization of acid

12

deuterium source is expected to offer convenient method with high deuterium incorporation

and high selectivity in the aromatic moiety.

1.2 Outline of the study

This study investigates synthesis of natural products and modification based on its

unique and enormous properties. Here in, direct halogenation of sucrose and 1-kestose, α-

amino acids extensive acylation, and hydrogen/deuterium exchange of cDOPA derivatives are

offering organic synthesis that utilizing chemical features of natural products become feasible.

The outline for this study was listed as below.

1.2.1 Direct halogenation at primary alcohols of carbohydrate

A. Direct halogenation at primary alcohols of sucrose

Tachrim, Z. P., et al., ChemistrySelect, 2016, 1, 58–63.

Regioselective halogenations of sucrose primary alcohols can simplify the synthesis of

carbohydrate-based products. The Appel reaction, using carbon tetrahalide (CBr4 or CCl4) and

triphenylphosphine, offers efficient conversion of primary hydroxy groups to halide. Previous

halogenation of sucrose with limited amounts of Appel reagent gave obscure result—

regarding the halogenation selectivity of sucrose primary alcohols at 6- and 6′-position.

Within careful purification of its per-O-acetylated form, resubjection of sucrose into Appel

reaction resulted in two mono- and one dihalogenated products only at 6- and/or 6′-position.

Extensive NMR analyses support the revision of assignment of previous literatures.

Comprehensive analysis of each monohalogenated sucrose at the primary positions, including

the first reported 1′-monohalogenated sucrose derivatives synthesis via regioselective enzymic

deacetylation, provided halogenation of sucrose primary alcohols by the Appel reaction

followed the order of 6>6′>>1′.

Regioselectivity order to form monohalogenation:

6>6'>>1'

6'>6>>1'6>6'>>1'

Previous Current

confuse

reactivity

Ambiguous NMR assignments

13

B. Direct halogenation at primary alcohols of 1-kestose

Tachrim, Z. P. et al, Arkivoc 2018, 341–348.

1-Kestose (O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl-(2→1)-α-D-

glucopyranoside) is a potential short chain fructooligosaccharide with an inulin-type skeleton.

Halogenation of 1-kestose was conducted via the Appel reaction with the use of carbon

tetrahalide (CBr4 or CCl4) and triphenylphosphine, which was then followed by conventional

acetylation. The per-O-acetylated form of 6,6′,6′′-trihalogenated derivatives of 1-kestose was

conveniently isolated. Subsequent deprotection of the per-O-acetylated form resulted in

yielding 6-, 6′-, and 6′′-trihalogenated derivatives. The structure elucidation by 1D- and 2D-

NMR established that halogenations are specific at the primary alcohols in the 6-, 6′-, and 6′′-

positions of the 1-kestose.

1.2.2 α-Amino acids extensive acylation

A. Friedel–Crafts Acylation of TFA-aliphatic -amino-N-hydroxysuccinimide ester

derivatives

Tachrim, Z. P., et al., Molecules, 2017, 22, 1748–1762.

Peptide bond formation

High storage stability

Easy purification

High reactivity

Previous

Study

Current Study

14

Chiral N-protected -amino aryl-ketones are one of the useful precursors used in the

synthesis of various biologically active compounds and can be constructed via Friedel–Crafts

acylation using N-protected -amino acids. One of the drawbacks of this reaction is the

utilization of toxic, corrosive and moisture-sensitive acylating reagents. In peptide

construction via amide bond formation, N-hydroxysuccinimide ester (OSu), which has high

storage stability, can react rapidly with amino components and produces fewer side reactions

than the acid chloride, including racemization. This study reports the first synthesis and

utilization of N-trifluoroacetyl (TFA)-protected -amino acid-OSu as a potential acyl donor

for Friedel–Crafts acylation into various arenes. The TFA-protected isoleucine derivative and

its diastereomer TFA-protected allo-isoleucine derivative were investigated to check the

retention of -proton chirality in the Friedel–Crafts reaction. Further utilization of OSu in

other branched-chain and unbranched-chain amino acids results in an adequate yield of TFA-

protected -amino aryl-ketone without loss of optical purity.

B. Friedel–Crafts Acylation of TFA-cyclic and aromatic -amino-N-hydroxysuccinimide

ester derivatives

Tachrim, Z. P., et al., Heterocycles, 2018, in press.

The utilization of N-trifluoroacetyl (TFA)-α-amino acid N-hydroxysuccinimide ester

(OSu) derivatives, a promising acylating agent with high storage stability, is reported for

Friedel–Crafts acylation into arenes and N-heterocycles. The reaction between TFA-Phe-OSu

derivatives and arenes introduces inter- and intra-molecular formation of C-acylation. TFA-

Tyr-OSu derivatives, which possess hydroxy substituent in the aromatic moiety of

phenylalanine, show specific inter-molecular acylation into benzene ring. The heterocyclic

TFA-Pro-OSu also shows relatively high reactivity toward acylation.

Intermolecular

Intramolecular

15

1.2.3 Hydrogen/deuterium exchange of cycloDOPA derivatives

Tachrim, Z. P. et al., Heterocycles, 2018, in press.

CycloDOPA (5,6-dihydroxy-indoline-2-carboxylic acid, leukodopachrome) is one of

metabolites derived from L-tyrosine, one of intermediate in melanin formation (mammalian)

and betanidin main skeleton (betalain pigment in plant). Synthesis of deuterated cDOPA via

hydrogen/deuterium exchange by utilization of deuterium chloride (DCl) and deuterated triflic

acid (TfOD) is reported. The novel fully deuterated aromatic cDOPA derivative can be

formed depending on temperature and time of H/D exchange condition. The complete study

of H/D exchange resulted in the selective deuterium between 4- and/or 7-position of aromatic

hydrogen of cDOPA.

16

Chapter 2 Direct halogenations at primary alcohols of carbohydrate

2.1 Introduction

Carbohydrates are bio-renewable resources fundamental to life, such as energy storage,

biological signaling, all recognition, and structural support in the plant kingdom. Common

sugars are available in large amounts from natural products and renewable source of starting

materials in organic synthesis. Among these carbohydrates, sucrose (Figure 1) is produced

worldwide in large quantities. This compound is highly functionalized molecules with

complex stereochemistry, due to the presence of several hydroxylated stereogenic centers.9

Sucrose is also accepted as a standard of sweet taste quality. Hence, the sweetness intensity

and other taste properties of natural and synthetic sweeteners are frequently compared with

sucrose.

The major problem faced for direct modification of cabohydrides is all of its hydroxy

groups having similar reactivity. Only by reaction within bulky reagents (e.g. halogenations),

it can be differentiated between the primary and secondary hydroxy groups. Sucrose, one of

the most widely used natural sweetener commercially available in large quantities, also faced

the same situation. Due to novelty, its chemical modification and transformation become high

potential for many purposes. In general, among primary alcohols of sucrose, the reactivity is

by the order of 6-OH and/or 6′-OH, and then least reactive neopentyl-like 1′-OH.51

The

halogenated sucrose analogues at the primary positions are known to be the most versatile

synthesis intermediates4,13,52,53

or for reversible contraceptive action in male rat.54

The

different sweetness activity is observed, if certain primary position of sucrose is halogenated.

For example, a commercial available artificial sweetener sucralose is introduced chlorination

on sucrose at the 4-, 1′- and 6′-positions on on the molecule enhanced 2000-fold sweetness

activity than sucrose (Table 1).3 Since these backgrounds, the selective halogenations,

particularly chlorination and bromination, only on primary hydroxy groups in sucrose have

been intensively studied in the last decades.

Various methods are available for halogenation on sucrose primary centers. Indirect

methods are included: the intermediate utilization;4,17–19

selective cleavage of fully protected

sucrose before further halogenation of the free primary alcohol counterpart;5–7

or bulky

functional groups protection and unprotection of one or two primary hydroxy groups.8–10

Procedures are also available, in which are allowed the direct replacement of primary hydroxy

groups with halogen, e.g. direct chlorination of sucrose by well-known sulphuryl chloride11

that gave high selectivity for monochlorination at 6′-position or the usage of other

17

reagents.13,14

However, the regioselectivity depends on the nature of the electrophilic reagent,

catalyst, or solvent used for the reaction. The controls of these variables are served more

efficiency rather than extensive protecting and deprotecting procedure which needed more

steps.7,8,55

Hence, the construction of the simple procedure will lead to great challenge for

beneficial desired product of direct modification on sucrose primary positions. Appel

reaction56

is one of convenient method for halogenations of primary alcohols by the use of

carbon tetrahalide (bromide or chloride) and triphenylphosphine (Figure 5 (a)). Appel reaction

also previously had been introduced into sucrose, to afford more efficient16

method for

halogenation of sucrose at its primary alcohols.

Figure 5 (a) Typical alcohol halogenation by Appel reaction; (b) Proposed mechanism of

Appel reaction on primary alcohol.

In 1978, an excess amount of Appel reagent was introduced and resulted in

dihalogenated sucrose at 6- and 6′-positions.15

Further modification of this reaction was

reported and observed that the mixture not only contained the diproducts but also two 6- and

6′-monohalo products, of which further purification and the separation of each isomer was

difficult.57

Many previous efforts15,55,58–60

by Appel reaction utilized different proportion and

condition for prior art of halogenations on sucrose primary alcohols at 6- and 6′-positions. By

commonly maintaining the ratio 2:1 of triphenylphosphine and carbon tetrahalide,

equivalency of these reagents to sucrose can be neglected if 6,6′-dihalogenated sucrose is the

desired product. Meanwhile, regioselectivity for 6- and 6′-monohalogenated sucrose

attainable by limiting the proportions of Appel reagents (e.g. carbon tetrahalide < 1.5 equiv.

from sucrose) is not completely identified. Previously, it has been shown61

that Appel reaction

is regioselective for the 6-position prior to 6′-position. In contrast, by limiting the proportions

18

of Appel reagents, 6′-monohalogented sucrose can be obtained in relatively high yield.9

Despite this success, re-subjection by similar reaction condition is remaining in the

preponderant product of 6-analogue as a mixture with 6′-halo,10

and no detailed consideration

has been performed for this observation.

Selective halogenation by limiting amount of Appel conditions is particularly

complicated, and separation of monoproduct from the mixture became another issue. For

decades, many studies have successfully halogenated sucrose at the primary positions,4–11,13–

15,17–19,55,58–61 but the reports describing properties of all former halodeoxysucrose derivatives

were limited, especially for the NMR analyses. NMR analyses are important to describe the

substitution site, but many reports are mainly focused the halogenation selectivity by

consideration on identical comparison of optical rotation with the previous literature data

which made the regioselectivity for sucrose as obscure due to unclear structural identifications

of each monohalogenated sucrose (especially its halomethylene portions). Therefore, the

needs to comprehensive analysis of all monohalogenated sucrose are essential. The focused

objective in this study is to re-subject sucrose into Appel reaction, by following previous

condition9

to afford the mono- and di-substituted products at 6- and/or 6′-position. The 1′-

monohalogenated sucrose was exclusively synthesized via chemoenzymatic reaction. Thus,

allowing the comprehensive study of structure elucidation by extensive NMR analyses of

halogenated sucroses at the primary positions, it was attempted to reveal the regioselectivity

of Appel reaction on sucrose primary alcohols. Moreover, 1′-kestose that elaborated with

additional fructose moiety at 1′-position was then used for checking the regioselectivity to

support the identification of the most hindered and less reactive position for halogenations

through Appel reaction. Full structural elucidation of halogenated sucrose and 1′-kestose

structure on its primary positions are performed.

2.2 Results and Discussion

Appel reaction, first reviewed by Rolf Appel,56

is a method to convert an alcohol to the

corresponding alkyl halide under mild conditions. By using triphenylphosphine and

carbontetrahalide (bromide or chloride) on the system, product can be observed in relatively

high yields. This reaction is somewhat similar to the Mitsunobu reaction, where the

combination of a phosphine, a diazo compound as a coupling reagent, and a nucleophile are

used to invert the stereochemistry of an alcohol or displace it. The reaction proceeds by

activation of the triphenylphosphine with the tetrahalomethane, followed by the attack of the

alcohol oxygen at phosphorus to generate an alkoxytriphenylphosphorane intermediate. The

19

oxygen is then transformed into a leaving group, and an SN2 displacement by halide takes

place, proceeding with inversion of configuration if the carbon is asymmetric (Figure 5 (b)).62

In this chapter, direct halogenations at primary alcohols (sucrose and 1-kestose) then trial to

subject to Appel reaction are described below.

2.2.1 Direct halogenations at primary alcohols of sucrose

A. Halogenation of sucrose via Appel reaction

Direct substitution of sucrose (1) with 1.2 equiv. carbon tetrabromide and 2.4 equiv.

triphenylphosphine at 60°C for 1.5 h,9 afforded complex mixture of several unprotected

halogenated sucrose derivatives. The separation of the mixture was complicated and

identification for each isomer by 1H NMR analysis was difficult due to observation of

overlapped signals, especially the modified primary centers. Even though number of

substituted carbon was readily distinguished by the upfiled location of their signals by 13

C

NMR, it is not easy to interpret the substitution site in this form. Most of the previous studies

on Appel reaction directly quenched these modified unprotected sucrose derivatives. In fact,

the dihalogenated product from sucrose was easily obtained, but our attempts to isolate the

monohalogenated products always remained as a mixture of a few isomers or contaminated

with the by-product;57

and no detail study was previously reported for this condition.

Scheme 3 Regioselective bromination of sucrose at the 6- and/or 6′-positions by Appel

reactions (2–4)

The effort to isolate and identify all brominated products was conducted by

conventional acetylation (Scheme 3) and found that the mixture consists of not only one9

major product in the TLC. When ethyl acetate or dichloromethane system was used, it did not

completely resolve the purification. Diethyl ether as the mobile phase was superior for the

isolation, not only to obtain the dibrominated product (7% yield), but also two

monobrominated products (7%–12% yield). Thus, pure compounds can be obtained for clear

NMR assignments. Separation between the two per-O-acetylated monohalogenated

20

regioisomers on silica gel column is a formidable endeavor. This term causes the NMR

assignment misinterpreted in previous study9 and indeed the overlapping signals that

associated with protons of several monoproducts10

can hamper the identification of

substitution site. In presents, four compounds were isolated from the mixture after column

chromatography: two compounds of per-O-acetylated monobrominated counterparts (2 and

3), one compound of per-O-acetylated dibrominated counterpart (4), and unreacted proportion

of sucrose octaacetate 5.

Each counterpart of per-O-acetylated halodeoxysucrose derivatives was subjected to 1D

NMR (1H and

13C) by the aid of 2D NMR (COSY, HETCOR, HMQC, HMBC, and NOESY),

along with optical rotation. The composition was confirmed by HRMS analysis. The detail

elucidation will be described in the next section. From the observation, the low isolated yield

for each counterpart is due to: (i) seting up an optimized condition for the highest yields of

each monohalogenated compound (or by increasing the molar ratio to promote more major di-

compound) and (ii) complete isolation of each component to measure with reliable NMR

analyses. However, Appel reaction can offer more efficient for direct modification on

unprotected sucrose rather than blocking one or two of the primary position with bulky

functional groups10

that needed more steps for the synthesis.

Limitation of NMR analysis for several decades leads to incomplete proton and carbon

signal assignments. Many studies mainly indentified the halogenated sucrose counterparts at

the primary position by only depending on comparison with optical rotation or transforming it

into the per-O-acetylated to facilitate the assignment by NMR analysis. Merely, their

identification still remained majorly as a multiplet for halogenated methylene. In the middle

of our approach to identify the monobrominated products, two main regions of the spectrum

are characterized in the 1H NMR. The proton resonances of aliphatic sugar groups were

observed in the downfield, and it can be distinguished from the halogenated methylene groups

that located in the upfield. As for NMR solvent, all the spin systems are more visible to be

conducted in CDCl3 ,59

rather than those in DMSO-d6.10

21

Figure 6 Selected 1H NMR (270 MHz, CDCl3) signals of brominated sucrose derivatives at 6-

and 6′-positions (2–4).

Compound 2 (Figure 6) showed a clear doublet at δH 3.62 ppm (J = 6.6 Hz)

corresponding to two protons of brominated methylene shown in the 1H NMR spectrum.

These spin systems are correlated with H-5′ on COSY assignment (Figure 7 (a)), and

ascertained the bromination position at C-6′. Multiplet assignment for H-5′ can be

differentiated by the COSY cross-peak between H-5′ and H-4′ and outer-space NOESY

correlation of H-5′ to H-3′. The halogenated terminal of C-6′ at δC 31.3 ppm is most proved by

HMBC technique with the observation of cross-peak with this carbon to H-4′. HETCOR and

HMQC coupling between the protons and carbon at 6′-position support this assignment for

recognition of the substitution site, which is supported by correlation of H-6′ with H-4′ and H-

5′. The differentiation between signals of protons and carbons on the glucose and fructose

rings with other positions, especially at the C-3, C-5, C-3′, C-4′, and C-5′, were confirmed by

HETCOR, HMQC and NOESY spectra. These assignments allowed the structure (gross) of 2

as per-O-acetylated 6′-bromo-6′-deoxysucrose heptaacetate.

4.50 4.00 3.50

δ 1H/ppm

H-6′

H-6a H-6b

H-6a H-6b

H-6′

H-1′

H-1′

H-6′

H-1′

H-6a H-6b

22

Figure 7 (a) Selected COSY (500 MHz, CDCl3) correlation of 6′-bromo-6′-deoxysucrose

heptaacetate (2); (b) selected COSY (500 MHz, CDCl3) correlation of 6-bromo-6-

deoxysucrose heptaacetate (3).

For 6-bromo-6-deoxysucrose heptaacetate (3), a typical pair of double-doublet at δH

3.42 and 3.60 ppm (J = 4.6, 11.5 Hz and J = 3.0, 11.5 Hz) are shown in 1H NMR (Figure 6).

The observation of the large geminal coupling for these spin systems can be distinguished in

COSY analyses by their correlation across the H-5 (Figure 7 (b)). Moreover, HMBC

correlation among these spin systems with C-4 supports the identification of bromination of

hydroxy groups at 6-position of sucrose. HETCOR and HMQC correlations of protons at 6-

position with C-6 (δC 31.1 ppm) also support this assignment, which confirmed by NOESY, to

elucidate compound 3 as the complete structure of 6-bromo-6-deoxysucrose heptaacetate.

Accordingly, spin systems on 1H NMR of 6- and 6′-monobrominated sucroses can be

differentiated. Based on alteration of these comprehensive assignments between 6- and 6′-

monobrominated sucroses, the previous study9 reported the misinterpreted NMR analysis of

6′-bromo-6′-deoxysucrose heptaacetate (2). In their report, 6′-bromo-6′-deoxysucrose

heptaacetate (2) was afforded a pair of double-doublet with the observation of geminal

coupling in 1H NMR. Hence, in this study, the assignment was revised from their previous

compound of 6′-bromo-6′-deoxysucrose heptaacetate (2) to 6-bromo-6-deoxysucrose

heptaacetate (3) and also refined new full description between these two monobrominated

products (2 and 3) to support our comprehensive structure recognition.

δ 1H/ppm

H-6′ H-6b

3.5

0 4

.00

4.5

0 5

.00

5.5

0

δ 1H

/pp

m

H-6′/H-5′H-4′/H-5′

H-1′

H-6a

COSY

3.5

0 4

.00

4.5

0 5

.00

5.5

0

δ 1H/p

pm

5.50 5.00 4.50 4.00 3.50

δ 1H/ppm

H-6a H-6b

H-1′H-6′

H-6a/H-5H-4/H-5

H-6b/H-5

COSY

5.50 5.00 4.50 4.00 3.50

(a) (b)

23

As for 6,6′-dibromo-6,6′-dideoxysucrose hexaacetate (4), splitting pattern in the upfield

region of 1H NMR is consisting of combination to those of the 6- and 6′-monobrominated

regioisomers (Figure 6). Appearance of a doublet at δH 3.65 ppm (J = 6.6 Hz) corresponding

to protons at 6′-position and double doublets at δH 3.52 and 3.40 ppm (J = 3.0, 11.5 Hz and J

= 5.6, 11.5 Hz) corresponding to each proton at 6-position that contributed geminal coupling

confirmed this dibromo analogue. Two primary carbons (C-6 and C-6′) were distinguished by

the upfield location of their signals. Two singlets at δC 31.5 ppm for C-6′ and δC 31.1 ppm for

C-6 are observed in 13

C NMR. Long range HMBC strictly distinguished carbon of this

position by the correlation between H-4 and C-6 for 6-position and H-4′ and C-6′ for 6′-

position. Our comparison of 6,6′-dibromo-6,6′-dideoxysucrose hexaacetate (4) in DMSO-d6 in

which specific region of the dibromomethyl was shown as a multiplet for protons at 6- and 6′-

position,10

made it difficult to identify each substituted position by 1H NMR spectroscopy. In

CDCl3, the splitting patterns of 6,6′-dibromo-6,6′-dideoxysucrose hexaacetate (4) for its

brominated methylene identification were visible, in line with the previous report.59

Moreover, the aid of COSY, HETCOR, HMQC, and NOESY constructed the complete

structure of 6,6′-dibromo-6,6′-dideoxysucrose hexaacetate (4).

The relative configurations of 2–4 were assigned in the basis of through-space NOESY

correlation. The anomeric carbon on all per-O-acetylated halodeoxysurose derivatives showed

α-1,2-glycosidic bond by the observation of NOESY correlation between H-1 and H-1′. As

for halogenation on 6-position, only one of the diastereotopic proton lies on the same face

with proton at 5-position by NOESY. Meanwhile, enantiotropic protons at 6′-position lies on

the same face which supported by NOESY correlation of these protons between H-4′ and H-

5′, respectively. Taken together with these correlations, C-6′ probably freely rotates but more

constrain in C-6. In line with these NOESY analyses, limitary intra-molecular hydrogen

bonding was only demonstrated on halogenated derivatives on 6′-position, thus shown by

large distance between H-6′ and its neighbor oxygen which observed by 3D modeling (Figure

8). Regulation of less steric orientation on brominated carbon that correlated to sucrose

conformation alteration builds up the unmerged geminal coupling constant, and specific

splitting pattern of protons on 6′-position then can be proposed.

24

Figure 8 Possible configuration of 6′-bromo-6′-deoxysucrose heptaacetate (2) viewed along

the fructofuranose ring. Dash line shows distance between O---H and arrow shows selected

NOESY correlation.

Similarly with previous bromination reaction, direct substitution of sucrose (1) with 1.2

equiv. carbon tetrachloride and 2.4 equiv. triphenylphosphine at 60°C for 1.5 h9 (Scheme 4)

was conducted. The direct quenching of modified unprotected sucrose derivatives that

contains a few isomers makes isolation of each compound difficult. Even though the

dihalogenated product from sucrose was easily obtained, each monochlorinated product which

was previously reported always as remained as a mixture10

needs endeavor purification

method. As mentioned in previously bromination of sucrose all of which halogenated product

can be isolate after conventional acetylation (Scheme 3), thus the attempt to isolate

chlorinated products was conducted by conventional acetylation (Scheme 4). When diethyl

ether as the mobile phase was used for the isolation by column chromatography, two

compounds of per-O-acetylated monohalogenated counterparts (6 and 7, 8%–13% yield), one

compound of per-O-acetylated dihalogenated counterpart (8, 5% yield), and unreacted

proportion of sucrose octaacetate 5 were found.

Scheme 4 Regioselective chlorination of sucrose at the 6- and/or 6′-positions by Appel

reactions (6–8)

25

Figure 9 Selected 1H NMR (270 MHz, CDCl3) signals of chlorinated sucrose derivatives at 6-

and 6′-positions (6–8).

With the same tendency, per-O-acetylated mono- and di-chlorinated products (6–8) also

showed a typical 1H NMR profile from those in the per-O-acetylated mono- and di-

brominated sucrose derivatives (Figure 9). Not only simplify the purification of

monochlorinated products from the mixture, but also per-O-acetylated monochlorinated

sucrose position of compounds 6 and 7 can be easily distinguished by 1H NMR. Chlorinated

position of compound 6 was identified by upfield spin system for protons at 6′-position

appearing as a doublet (J = 6.6 Hz) at δH 3.77 ppm (Figure 10 (a)). Different splitting pattern

happened for 6-chloro-6-deoxysucrose heptaacetate (7) which contributed to geminal

coupling for each spin system at 6-position was shown as a set of double-doublets (J = 4.6,

12.2 Hz and J = 3.0, 12.2 Hz) at δH 3.58 and 3.73 ppm (Figure 10 (b)). Then, compound 8 was

categorized as 6,6′-dichloro hexaacetate which appeared as a doublet (J = 6.3 Hz) at δH 3.79

ppm toward protons at 6′-position and the observation of geminal coupling for each

diastereotopic proton at 6-position which assigned as a set of double doublets (J = 4.9, 12.2

Hz and J = 3.0, 12.2 Hz) at δH 3.56 and 3.67 ppm.

δ 1H/ppm

6

7

8

H-6′

H-6a H-6b

H-6a H-6b

H-6′

H-1′

H-1′ H-6′

H-1′

H-6a H-6b

26

Figure 10 (a) Selected COSY (500 MHz, CDCl3) correlation of 6′-chloro-6′-deoxysucrose

heptaacetate (6); (b) selected COSY (500 MHz, CDCl3) correlation of 6-chloro-6-

deoxysucrose heptaacetate (7)

In line with brominated products, chlorinated position of compounds 6–8 was also

shown by 13

C NMR spectroscopy which allowed the C-6 and C-6′ primary carbon on upfield

region for chlorinated alkyl signals to be distinguished. Monochlorinated sucrose can be

easily identified by appearance of singlet at δC 43.9 ppm belongs to C-6′ for 6′-chloro-6′-

deoxysucrose heptaacetate (6) and δC 43.0 ppm belongs to C-6 for 6-chloro-6-deoxysucrose

heptaacetate (7) in 13

C NMR. Compound 8 showed two singlets at δC 44.0 ppm assigned for

6-position and δC 43.2 ppm assigned for 6′-position. The gross structure of two

monochlorinated derivatives 6 and 7 and also dichlorinated derivative 8 were helped by the

aid of COSY, HETCOR and HMQC. Furthermore, HMBC and NOESY correlations most

proved the chlorinated site of these compounds.

To meet our purpose, acetylation was preferred to protect the halodeoxysucrose

derivatives rather than other protection groups due to its more offered convenience for the

separation and structural elucidation. It is also common opinion in many literatures that

acetylation of the sugar derivatives facilitates and simplifies the NMR analysis.63

However, up

to date, detail of structure characterization for per-O-acetylated halodeoxysucrose derivatives

at 6- or 6′-position (2–4 and 6–8) is not completely identified. Indeed, the 1H NMR

H-6a H-6b

H-1′

H-6′

H-1′

H-6a

H-6b

H-6′

H-6′/H-5′

H-4′/H-5′

δ 1H/ppm

δ 1H/p

pm

δ 1H

/pp

m

δ 1H/ppm

6a-H/5-H

4-H/5-H

6b-H/5-H

COSY COSY

(a) (b)

27

assignment of proton at 6- and 6′-position has been reported as multiplets10

and hampered the

substitution site recognition for substitution. Comparison with the literature data for all

compounds also described for supporting our comprehensive structural elucidation (see Table

3–8 for detail previous literature data comparison). Furthermore, our achievement for

isolation of per-O-acetylated monohalogenated sucrose derivatives (2, 3, 6 and 7), by using

silica gel column with diethyl ether system, completely resolved the 1H NMR assignment.

The multiplicity of overlapping protons of halomethylene moiety at 6- or 6′-position on

previous report10

can be identified by the observation of distinguishable signals with specific

splitting pattern in the upfield region of 1H NMR.

In Appel reaction, monohalosubstituted sucroses at the 6-position (3 and 7) emerged as

the major products with yields of 12% and 13%, respectively. Based on these results, the

regioselectivity for monohalogenation (bromination and chlorination) of unprotected sucrose

by using limited proportions of Appel reaction followed the order 6>6′>>1′. The mechanism

of Appel reaction involved formation of alkoxytriphenylphosphorane intermediates.56

The

regioselectivity was observed in this reaction presumably because of nucleophilic

displacement of triphenylphosphine oxide by halide anion, which preferred 6-position rather

than 6′-position. In the basis of the solution conformations of sucrose in aprotic solvents such

as pyridine,64

the 1′-position is the type of neopentyl-like, more hindered position, and

considerably less reactive due to a bulky nucleophile under mild conditions of Appel

reaction.63,64

In the case of chlorination, selective displacement required a large excess of

CCl4 due to its volatility.10

Moreover, the second substitution might occur in slow manner,

therefore moderate yields of the diproducts 4 and 8 could be observed.

B. Chemoenzymatic synthesis of 1′-monohalogenated sucrose

Our comprehensive NMR analysis revealed that the use of limited proportion of Appel

reagents resulted in unsubstituted hydroxy groups either at secondary position or the most

hindered 1′-position. Based on NMR analysis (Figures 6, 7, 9, and 10), monohalogenated

sucrose derivates at 6- or 6′-position can be distinguished. Multiplicity for protons at 6′-

position of 6′-monohalo 2 and 6 are observed without geminal coupling at 1H NMR.

Theoretically, this term belongs to protons at 1′-position of 1′-monohalogenated sucrose

counterpart (10 or 11). To ensure these results, the structure analysis was continued to

exclusive synthesis of 1′-monohalgenated sucrose. The attempt to halogenate 1′-position was

studied via chemoenzymatic reaction (Scheme 5).6,65–67

From the selective enzymatic

hydrolysis of 5 previously reported, several enzymes are available in cleavage at the 1′-acetyl

group. Alcalase seems suitable for this purpose due to its shorter reaction time and higher

28

yield of sucrose heptaacetate (9) as the major product.65

By the partly acetylated sucrose,

2,3,4,6,3′,4′-hexa-O-acetyl-sucrose,6,68

the free hydroxy group at 1′-position was not reactive

with Appel reagents. Accordingly, compound 9 was then triflouromethanesulfonylated and

halogenated by using lithium halides (bromide and chloride) to give 1′-bromo-1′-

deoxysucrose heptaacetate (10) for bromination and 1′-chloro-1′-deoxysucrose heptaacetate

(11) for chlorination. However, brominated chemoenzymatic product of 10 and its

deacetylated counterpart (20) is firstly reported in this study (see Tables 9 and 10 for previous

literature data comparison).

Scheme 5 Chemoenzymatic synthesis of 1′-monohalogenated sucrose derivatives (10 and

11)6,65–67

The assignment of the regioisomeric halogenated sucrose 1′-bromo-1′-deoxysucrose

heptaacetate (10) can be verified by a distinct pair of doublets with a large geminal coupling

constant (J = 12.0 Hz) at δH 3.61 and 3.49 ppm assigned for protons at 1′-position in the 1H

NMR (Figure 11). NOESY correlation of these spin systems with proton at 1-position showed

that bromination was conducted near the anomeric carbon. By the assignments of protons that

showing a long range coupling at 1′-position to C-2′ on HMBC, along with the HMQC

correlation to unsure these protons associated on the same carbon, this particular spin-system

of monobrominated at 1′-position can be differentiated from 6- and 6′-bromo sucroses (Figure

12). Next, the assignment of 1′-chloro-1′-deoxysucrose heptaacetate (11) is similar to those in

1′-bromo-1′-deoxysucrose heptaacetate (10) in which a pair of germinal coupled methylene

protons at δH 3.74 and 3.57 ppm (J = 12.0 Hz) is clearly defined for each of proton signals in

this counterpart.

29

Figure 11 Selected 1H NMR (500 MHz, CDCl3) signals of 1′-bromo-1′-deoxysucrose

heptaacetate (10)

Figure 12 Selected COSY, HMQC, and HMBC (500 MHz, CDCl3) correlation of 1′-bromo-

1′-deoxysucrose heptaacetate (10).

On 13

C NMR, the upfield signal at δC 33.5 ppm was assigned for 1′-bromo-1′-

deoxysucrose heptaacetate (10) and δC 45.3 ppm was assigned for 1′-chloro-1′-deoxysucrose

heptaacetate (11). Long-ranged correlation on HMBC between H-3′ with C-1′ was a strong

evidences for supporting this assignment (Figure 12). The gross structure of per-O-acetylated

halodeoxysucrose on 1′-position was elucidated by COSY, HETCOR, HMQC, and HMBC.

Through-space NOESY correlation indicated relative configuration of per-O-acetylated 1′-

halodeoxysucrose derivatives 10 and 11 where this halogenated methylene near the anomeric

1′a-H 1′b-H

4.50 4.00 3.50δ 1H/ppm

6b-H

6′-H

6a-H

3.4

0

3.8

0

δ 1H/p

pm

30

3

53

0

35

δ 13C

/pp

mδ 1

3C/p

pm

6.00 5.50 5.00 4.50 4.00 3.50 3.00

δ 1H/ppm

3′-H/ C-1′

1′a-H/C-1′

1′b-H/C-1′

1′a-H/1′b-H

1′a-H 1′b-H

6a-H

6′-H 6b-H

COSY

HMQC

HMBC

30

position α-1,2-glycosidic bond can be distinguished. NOESY correlation of H-1′a and H-1′b

to H-1 indicated 1H NMR splitting pattern in conjunction with a large geminal coupling (J =

12.0 Hz) exemplify different spin system of each proton on halogenated 1′-position. In

contrast to previous explanation of monohalogenated at 6- or 6′-position of 2, 3, 6, and 7,

geminal coupling observed might be caused by intra-molecular hydrogen-bonding between

the electron lone pair of the neighbor oxygen with the proton on this position. As an example,

representation of small distance between protons at 1′-position and oxygen on

fructopyranoside unit (2.3 Å) of 1′-bromo-1′-deoxysucrose heptaacetate (10) was observed by

3D models.

C. Comprehensive comparison of halogenated sucrose with per-O-acetylated

commercial available sucralose

Sucralose (12) is a commercially available artificial sweetener produced from sucrose

which promoted chlorination on 4-, 1′- and 6′-positions of the sugar molecule. For complete

assignment of comprehensive study on halogenated sucrose at the primary position, sucralose

(12) was then protected by conventional acetylation and additionally assigned to NMR

analysis. By the form of per-O-acetylated sucralose (13, Scheme 6), known as TOSPA

(trichlorogalactosucrose pentaacetate), chlorinated tertiary carbon at the 4-position was

located in the middle field of 1H NMR spectrum (Figure 13). Thus, it made the chlorinated

carbon at its halogenated primary center at 1′-, and 6′-positions easily identify by 1H NMR.

Specific coupling and splitting pattern of the chloromethyl proton at this position can be

observed in the upfield region. A fair doublet (J = 5.7 Hz) at δH 3.77 ppm corresponding to

protons at 6′-position is differentiated from a pair of doublets (J = 12.0 Hz) at δH 3.60 and

3.71 ppm corresponding to each proton at 1′-position. Furthermore, tertiary carbon at the 4-

position was located in slightly middle field of 13

C NMR spectrum and allowing the two

specific signals on upfield region at δC 44.4 and 43.8 ppm to be briefly differentiate the 1′-

and 6′-positions.

Scheme 6 Structure of sucralose (12) and sucralose pentaacetate (13)

31

Cross-peak between H-6′ to H-5′ on COSY distinguished the fine doublet at the upfield

region as chloromethylene at 6′-position. NOESY correlation H-6′ and H-4′ and also H-5′

strictly differentiated this assignment. Furthermore, the two primary chlorinated carbons are

utmost elucidated by HMBC with the correlation from H-3′ to C-1′ and from H-4′ to C-6′

which all these spin systems are supported by HETCOR and HMQC. All of these assignments

for the upfiled spin system on 1H and

13C NMR which also supported by 2D NMR, allowing

brief structural analysis thus compared 6 with 11, which showed the same tendency, in line

with the sucralose pentaacetate (13).

Figure 13 Selected 1H-NMR (500 MHz, CDCl3) of sucralose pentaacetate (13)

D. Deacetylation of per-O-acetylated halogenated sucrose

Complete structural elucidation of regioselective per-O-acetylated monohalogenated (2,

3, 6, 7, 10 and 11) and dihalogenated sucrose derivatives (4 and 8) was conducted by

deprotection (Scheme 7) and also assigned for the precise NMR analyses. Due to complex

mixtures were afforded by sodium methoxide, deacetylation was conducted using saturated

ammonia in methanol. Structure elucidation of all regioisomeric halogenated sucrose

derivatives, in particular for identification among the protons of the three possible substitution

site at 6-, 1′- and 6′-position, was complicated because of several overlapping 1H NMR

signals. It is not easy to distinguish the sample that was contained small amounts of by-

products on 1H NMR for this deacetylated form. However, the per-O-acetylated form is

advance for clear structure construction since its specific region and splitting pattern of the 1H

NMR spectra simply identify halogenated position rather than directly assigned the

unprotected form.

H-6′

H-1′a H-1′b

H-4, H-5 H-6, H-5′

32

Scheme 7 Deacetylation of per-O-acetylated halodeoxysucrose derivatives (2–4, 6–8, 10, and

11).

In the case of structure elucidation of sucralose (12, Scheme 6), most of chlorinated

positions can be distinguished easily from other positions. The spin systems of protons at 4-,

1′- and 6′-positions in 1H NMR are shown as a specific splitting pattern at δH 4.49, 3.76 and

3.87 ppm, respectively. Meanwhile, all spin systems of regioisomeric halogenated sucrose

derivatives are located in relatively middle field, of which protons on the sugar moiety (not

only from halogenated methylene) appeared within same region and cause signal overlapping

that is difficult to identify. Commonly, differentiation within the 1H NMR specific region for

modified unprotected sucrose is performed by comparison with sucrose. It has also been

studied,12

that the 13

C NMR assignment of the halogenated primary carbons (C-6, C-1′, and C-

6′) can be tentatively predicted by the comparison with sucrose and galacto-sucrose based on

epimerisation at C-4 chemical shift. However, although the halogenated counterparts are

readily distinguished by the upfield location of their signals in 1H and

13C NMR,

differentiation for the substitution site is still unsolved. In agreement, many previous studies

elucidated the modified counterpart of halogenated unprotected sucrose by subjection into

HMBC analysis.69

In this study, the integration of several 2D NMR analyses (COSY,

HETCOR, HMQC, HMBC, NOESY, and TOCSY) is needed for brief overlapping signal

assignments to distinguish the substitution site and also to fully construct the gross structure

of each unprotected halogenated sucrose derivative (14–21).

33

Figure 14 1H NMR signals (500 MHz, D2O) of (a) 6′-bromo-6′-deoxysucrose (14); (b) 6-

bromo-6-deoxysucrose (15); (c) 6,6′-dibromo-6,6′-deoxysucrose (16); (d) 1′-bromo-1′-

deoxysucrose (20).

Deacetylation on bromodeoxysucrose derivatives 2–4 and 10 gave bromodeoxysucrose

derivatives 14–16 and 20. Firstly, in 1H NMR of 6′-bromo-6′-deoxysucrose (14) (Figure 14

(a)), protons at 6′-position were shown as a multiplet at δH 3.73–3.66 ppm, and thus by COSY

correlation between H-6′ and H-5′ and close assignment by TOCSY correlation betweem H-6′

and H-3′ and H-6′ and H-5′, differentiation from protons at 6-position can be determined. The

confirmation can also be seen by the cross-peak between H-4′ and C-6′ on HMBC. The

overlapping 1H NMR signals in the middle field are strictly differentiated by HMQC to

identify protons at 6′-position. NOESY correlation between H-6′ and H-4′ also supported

these assignments. Up to date, structural elucidation of compound 14 was hampered due to

difficulties on its isolation—always remained as a mixture with the 6-halo derivatives.10,57

Herein, detailed analyses of the first purified 6′-bromo-6′-deoxysucrose (14) was stated and

even assigned 6-bromo-6-deoxysucrose (15) (including the monochlorinated products 17 and

H-1′

H-6b

H-6a

H-6′H-3′

H-3′

H-3′ H-1′

H-6b

H-6′H-6a

H-1′H-6

H-6′

H-3′ H-1′b

H-6

H-6′H-1′a

(a)

(b)

(c)

(d)

14

15

16

20

Overlap region

5.50 4.50 4.00 3.50

δ 1H/ppm

34

18, as described leter) to clearly distinguished its structure. Next, monobrominated sucrose at

6-position of 6-bromo-6-deoxysucrose (15) can be clearly differentiated from 6′-bromo-6′-

deoxysucrose (14) by the observation of multiplet at δH 3.76–3.71 ppm of H-6 (Figure 14 (b)).

These spin systems can be differentiated from other protons only by the cross peak between

H-6 and H-5 on TOCSY and HMBC correlation from H-6 to C-4. Complete positional

assignment for gross structure of 14 and 15 was supported by COSY, HETCOR, and HMQC.

It was difficult to assign the substituted position of 6,6′-dibromo-6,6′-dideoxysucrose

(16) by 1H NMR (Figure 14 (c)) where protons on 6- or 6′-brominated position are overlapped

with other protons and appeared as a multiplet at δH 3.75–3.69 ppm for protons at 6′-position,

at δH 3.63–3.60 and 3.69–3.65 ppm for protons at 6-position. Structure elucidation of 6,6′-

dibromo-6,6′-dideoxysucrose (16) by TOCSY showed the correlation of H-6′ with H-3′, H-4′,

and H-5′, allowing fructo-configuration of protons at 6′-position. Then, by through-space

correlation NOESY, 6,6′-dibromo-6,6′-dideoxysucrose (16) showed that diastereotopic proton

H-6b was in the same face with H-5 which spin system at 6-position to be assigned. The last,

assignment for bromination on hindered 1′-bromo-1′-deoxysucrose (20) showed alteration

between multiplet at δH 3.73–3.69 ppm for 1′a-H and broad singlet at 3.63 ppm for for H-1′b

(Figure 14 (d)). Relative configuration of 20 by NOESY correlation showed 1′a-H was in the

same face with H-1, signified this bromination splitting pattern on fructo-configuration. Large

coupling constant on 1′-bromo-1′-deoxysucrose (20) can be observed by correlation of 1′a-H

and H-1′b to C-2′ and C-3′on HMBC.

The de-O-acetylated bromodeoxysucroses 14–16 and 20 are also assigned by 13

C NMR.

In the case of monobrominated derivatives, 13

C NMR upfield region is shown as a singlet at

δC 33.2 ppm that belongs to C-6′ for compound 14 and δC 33.4 to C-6 for compound 15. On

long range HMBC, 3JH-C correlation from H-4′ to C-6′ for compound 14 and

3JH-C correlation

from H-4 to C-6 for compound 15 also confirmed position of monobromination specific on 6′-

or 6-position. Complicated assignment was found in 13

C NMR spectrum of compound 16, as

the coincide signals at the upfield region was difficult to elucidate. These signals can be

critically elucidated only by HMBC in the appearance of the cross peak between H-4 and C-6

and also H-4′ and C-6′ in which δC 33.3 ppm is assigned as C-6, while δC 33.2 ppm is

assigned as C-6′. Next, the 13

C NMR spectrum of 1′-bromo-1′-deoxysucrose (20) showed

singlet δC 32.6 ppm to confirm the bromination at this anomeric position. Cross peak of H-3′

with upfield singlet of C-1′ in HMBC spectrum supported bromination on the anomeric

position. In line with these assignments, COSY, HETCOR, HMQC, and NOESY also allowed

full structure construction of all de-O-acetylated bromodeoxysucroses 14–16 and 20.

35

Figure 15 1H NMR signals (500 MHz, D2O) of (a) 6′-chloro-6′-deoxysucrose (17); (b) 6-

bromo-6-deoxysucrose (18); (c) 6,6′-dibromo-6,6′-deoxysucrose (19); (d) 1′-bromo-1′-

deoxysucrose (21).

Deacetylations on per-O-acetylated chlorodeoxysucrose derivatives 6–8 and 11 resulted

in yielding chlorodeoxysucrose derivatives 17–19 and 21, respectively, which had the same

tendencies with the previous formations of de-O-acetylated bromodeoxysucrose derivatives

14–16 and 20. From the monochlorinated products, multiplet at δH 3.85–3.77 ppm in 1H NMR

(Figure 15 (a)) spectrum is assigned for compound 17 which was distinguished as chlorinated

sucrose only at 6′-position center. Differentiation by cross peak of H-6′ with H-5′ on COSY

and supported by TOCSY and NOESY cross peaks between H-6′ and H-3′, H-6′ and H-4′, and

also H-6′ and H-5′ supported identification of this spin system. Meanwhile, compound 18

appeared as multiplet at δH 3.86–3.82 ppm for chlorination at 6-position (Figure 15 (b)).

These multiplicities were supported by correlation between H-6 and H-5 on TOCSY that

assigned chlorination only on the glucose moiety.

H-1′ H-3′

H-3′

H-3′

H-1′ H-6bH-6a

H-6′

H-1′H-6 H-6′

H-3′

H-1′H-6

H-6′

(a)

(b)

(c)

(d)

17

18

19

21

Overlap region

δ 1H/ppm

H-6 H-6′

36

Among the chlorinated sucrose derivatives, substituted site of 6,6′-dichloro-6,6′-

dideoxysucrose (19) is the most difficult to distinguished by 1H NMR (Figure 15 (c)), which

appeared as multiplet at δH 3.84–3.78 ppm assigned for H-6′ and at δH 3.91–3.84 ppm

assigned for H-6. Only by alteration within TOCSY correlation between H-6′ and H-3′ / H-4′ /

H-5′ can differentiate chlorinated position on the fructose and glucose moiety. HMBC

correlation from H-6′ to C-4′ and C-5′ and from H-6 to C-3 and C-4 was fully consistent with

this assignment. Then, as for 1′-chloro-1′-deoxysucrose (21), 1H NMR (Figure 15 (d)) showed

doublet at δH 3.76 ppm (J = 4.0 Hz) which showed correlation on TOCSY within the cross

peak between H-1′ and H-4′. Outer-space correlation of NOESY also allowed this splitting

pattern to assign proton on H-1′ was close to H-1 indicating chlorination near the anomeric

position. Strong evidence was presented by long ranged correlation HMBC that exhibited the

cross peaks from H-1′ to C-2′ and C-3′.

As for 13

C NMR, the chlorination assignment of compound 17 at 6′-position can be

performed by a distinguishable upfield singlet on 13

C NMR at δC 45.1 ppm that was also

supported by correlation with H-4′ on HMBC. As for 6-chloro-6-deoxysucrose (18), cross

peak of C-6 (δC 44.2 ppm) on HMBC with H-4 gave a strong evidence for this assignment.

Next, two singlets on upfiled region of 6,6′-dichloro-6,6′-dideoxysucrose (19) at δC 45.2 and

44.3 ppm are assigned for C-6′ and C-6, repectively. These signals are supported by the cross

peak from C-6′ to H-4′ and to H-5′ and C-6 to H-4 on HMBC, thus chlorination allowed on 6′-

and 6-position was deduced. Last, singlet of C-1′ at δC 43.8 ppm on the upfield region of 1′-

chloro-1′-deoxysucrose (21) 13

C NMR confirmed the substitution occurred only at 1′-position.

HETCOR and HMQC supported all of these assignments for the gross structures of 17–19

and 21.

Bhattacharjee and Mayer57

reported the detail experimental study (retention time of the

TLC separation, but without full description of NMR analysis) for unprotected halogenated

sucrose that was synthesized by using an excess amount of Appel reagents. In this report, the

attempt to isolate monohalogenated products (unprotected sucrose form) always remained as

mixture—monochlorinated sucrose required more complex purification process—and they

cannot solve these purification problems. Similar with this report, in 2011, Barros et al.10

stated that mixtures of 6- and 6′-substituted sucrose were obtained for chlorination products, if

limited proportion of Appel reagents were applied for sucrose. In this study, purification was

conducted by the per-O-acetylated form, and after identification of each purified isomer, the

de-O-acetylated was conducted. Therefore, pure de-O-acetylated modified sucrose at primary

counterpart (14–21) can be identified. Complete structural elucidation of this study also

compared with the previous literature data (see Table 13–19). However, structures and

37

substitution sites of all the unprotected halogenated sucrose derivatives (14–21) were

consistent with those in per-O-acetylated forms (2–4, 6–8, 10, and 11).

2.2.2 Direct halogenations at primary alcohols of 1-kestose

A. Halogenation of 1-kestose by Appel reaction

Inulin-type short-chain fructooligosaccharides (FOS) are fructose oligomers that consist

of a terminal glucosyl unit and two to five fructosyl units. They are recognized as prebiotic

indigestible organosaccharides70

and due to this property, demand of FOS has been increasing

in the food industry.23

1-Kestose (22, Figure 16) is one of FOS that has a β-D-fructofuranosyl

group on O-1 of the D-fructosyl moiety of sucrose (1, Scheme 3). 1-Kestose 22 is naturally

found in honey and some plants belonging to the Amaryllidaceae family.71–73

The interest in

1-kestose 22, as a low-calorie food ingredient, continues to increase due to its sweetening

power. FOS syrup enriched by 1-kestose 22 can be used as an alternative sweetener for

diabetics.24

To produce 1-kestose 22, the enzyme derived from the leaves of sugar beets have

demonstrated transfructosylation activity in the present of sucrose 1; thus, the products of

transfer ring reaction were mainly 1-kestose 22 with smaller proportions of other

oligosaccharides.74

Commercial cellulolytic enzymes have also been studied for preparation

of FOS with high 1-kestose concentrations (around 68.2%, calculated as short-chain FOS).23

Figure 16 Structure of 1-kestose (22) and undecaacetate of 1-kestose (23).

The simple direct halogenations of carbohydrate become one of the interest due to the

previously reported study, revealing that specific substitution of sucrose primary hydroxy

groups by chloride can enhance the sweetness activity,3 and can potentially be used as an

alternative sweetener. As previously mentioned, halogenations by utilization of Appel

reaction are one of the convenient methods to convert the primary hydroxy group into a halo

methylene group in the presence of triphenylphosphine and carbon tetrahalide.56

Halogenation

(particularly for chlorination and bromination) of primary alcohols of sucrose 151

—the main

precursor for enzyme-catalyst generation of 1-kestose 22—has been studied since the 1970s,15

but the reactivity of primary hydroxy groups attained by the Appel reaction has not been

38

completely identified due to limitation of structure analysis. As mentioned in previous

explanation, sucrose 1 can be selectively brominated and chlorinated using the Appel reaction

at only the 6- and 6′- positions with no halogenation at 1′-position supported by one- and two-

dimensional (1D and 2D, respectively) NMR analyses (Schemes 3 and 4).75

As for trisaccharide modification, halogenation of the primary hydroxy groups of

raffinose (O-α-D-galactopyranosyl-(1→6)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside)

has been reported previously.20

Raffinose reacted with sulfuryl chloride and the authors have

found that the chlorinated proportion at the primary positions was followed by chlorination on

secondary alcohols at the 4-position of the galactopyranosyl moiety. To date, no study has

been attempted to substitute primary alcohols of FOS using halogens. In midst of FOS, 1-

kestose 22 has shown relatively high sweetness activity and is already commercially available.

Therefore, modification of its primary alcohol would potentially increase the synthetic

opportunity for alternative sweeteners. The aimed of this study is to synthesize halogenated 1-

kestose 22 at the primary positions by using the Appel reaction to comprehensively study the

structure elucidation supported by 1D and 2D NMR analyses.

Direct substitution of 1-kestose 22 with 4.7 equiv. carbon tetrahalide (bromide or

chloride) and 9.1 equiv. triphenylphosphine at 70 oC for two hours (Scheme 8) produced a

complex mixture with complicated 1H-NMR spectrum due to observation of overlap signals,

especially the modified primary centers. The mixture was separated after conventional

acetylation. Further purification was conducted using an ether and hexane system as the

representative mobile phase to isolate halogenated carbohydrate. The ethyl acetate or

dichloromethane system cannot be used for the halogenated proportion.75

The mixture mainly

yielded halogenated 1-kestose derivatives in the pre-O-acetylated form (bromination 24 and

chlorination 25, Scheme 8).

Scheme 8 Halogenation of 1-kestose (22) via Appel reaction with Ph3P and carbon tetrahalide

to produce pre-O-acetylated form of halogenated 1-kestose derivatives (bromination 24 and

chlorination 25).

39

The 1H NMR analyses for peracetylated 24 and 25 showed two regions that contribute

to the proton resonance of aliphatic sugar groups in the downfield region and halogenated

methylene groups located in the upfield region. Based on 13

C NMR, three halogenated

methylene carbons in the upfield region were easily determined in the halogenated proportion

in the 1-kestose derivatives, but the halogenated position remained unclear. Therefore, 2D

NMR (COSY, HETCOR, HMQC, HMBC, NOESY, and TOCSY) analyses were used to

determine the halogenated position. For this purpose, CDCl359,75

was used as the solvent to

increase the visibility of the spin system during the assignment. Undecaacetate 1-kestose71,76

(23, Figure 16, see Table 20 in experiment section for 1D and 2D NMR and comparison of

structure elucidation with the literature) was identified and used to understand the sugar

skeleton of halogenated 1-kestose derivatives.

During investigation of brominated position on the 1-kestose derivative, 1H-NMR of

compound 24 showed eight proton signals in the upfield region. The excess of two protons

corresponds to the H-1′ glycosidic bond of O-β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl,

which showed a considerable geminal coupling with a typical pair of doublets at δH 3.79 and

3.73 ppm (J = 10.3 Hz). The correlation between H-1′a and H-1′b with C-2′ in HMBC,

supported by HETCOR and HMQC, distinguished this spin system. The same tendency was

also demonstrated by the undecaacetate of 1-kestose 23, which supported the glycosidic bond

signals. The additional six proton signals in the upfield region highlighted the specific

brominated proportion of compound 24. A clear pair of doublets at δH 3.54 and 3.40 ppm (J =

2.3 and 11.5 Hz and J = 6.3 and 11.5 Hz, respectively) were observed by 1H-NMR. The

geminal coupling for these spin systems indicated the brominated methylene of the glucose

ring at the 6-position, which was easily distinguished by its correlation with H-5 in COSY and

with H-4 and H-5 in TOCSY (Figure 17 (a)). Moreover, the carbon brominated terminal at the

6-position (δC 31.1 ppm) was also identified, as supported by HMBC analyses. The

observation of the long range correlation between C-6 and H-4 clearly distinguished

brominated position which supported by HMQC and HETCOR. The protons and carbons

signals of the glucose ring were distinguished from signals of fructose rings, especially at the

C-3, C-4, and C-5 positions, using TOCSY and NOESY, which were also supported by

HETCOR, HMQC, and HMBC analyses.

Among fructose rings of 1-kestose, there are three possible primary alcohols at the 6′-,

1′′-, and 6′′-positions that are readily brominated. The upfield region of 1H-NMR typically

showed two types of doublets at δH 3.66 ppm (J = 6.9 Hz) and 3.69 ppm (J = 7.4 Hz),

indicating that bromination occurred only at the 6′- and 6′′-positions of compound 24,

respectively. The correlation between H-6′ and H-5′ in COSY and between H-6′ and H-4′ / H-

40

5′ in TOCSY differentiated the bromination at the 6′-position (Figure 17 (a)). Similarly,

brominated 6′′-position of compound 24 showed these particular correlations. The brominated

terminals of C-6′ at δC 32.0 ppm and C-6′′ at δC 32.7 ppm were determined mostly using

HMBC with the observation of a cross peak of these carbons at H-4′ and H-4′′, respectively.

HETCOR and HMQC coupling between the protons and carbon at the 6′- and/or 6′′-position

supported the identification of the bromination position.

The aliphatic sugar groups between the two fructose rings of compound 24 were

differentiated using TOCSY and NOESY, of which assignment was also supported by COSY,

HETCOR, HMQC, and HMBC analyses. Based on 1H- and

13C-NMR and support of 2D

NMR analysis, the 1′′-position of compound 24 showed no bromination. The 1′′-position of

the fructose ring is known to be a neopentyl-like51,75

terminal having the least reactive

proportion. This phenomenon also occurred when sucrose was used.75

According to these

approaches, the bromination product of 1-kestose was determined to be 1′′,2,3,3′,3′′,4,4′,4′′-

octa-O-acetyl-6,6′,6′′-tribromo-6,6′,6′′-trideoxy-1-kestose (24).

For the chlorination product of 25, the spin system in 1H-NMR showed eight proton

signals in the upfield region. Unlike the previous bromination product 24 or undecaacetate 1-

kestose 23, the H-1′ of compound 25 exhibited no geminal coupling and presented as a singlet

at δH 3.75 ppm supported by HMBC, HETCOR, and HMQC. The chlorinated proportion at

the 6-position was shown as a pair of double doublets at δH 3.68 and 3.57 ppm (J = 2.3, 12.0

Hz and J = 5.7, 12.0 Hz, respectively). A clear correlation of this chlorinated methylene of the

glucose ring with H-5 in COSY and H-4 and H-5 in TOCSY was observed (Figure 17 (b)). As

for the 1H-NMR spin system of 6′- and 6′′-chloro of compound 25, the overlapped signals at

δH 3.82–3.78 ppm demonstrated its correlation with H-5′ and H-5′′ in COSY and with H-4′,

H-5′, H-4′′, and H-5′′ in TOCSY. The brominated terminal of C-6′ at δC 44.4 ppm and C-6′′ at

δC 44.6 ppm highlighted the correlated cross peaks of these carbons to H-4′ and H-4′′,

respectively. HMBC strongly distinguished the chlorinated 6′- and 6′′-positions of compound

25. These results support that the chlorinated product of 1-kestose is 1′′,2,3,3′,3′′,4,4′,4′′-octa-

O-acetyl-6,6′,6′′-trichloro-6,6′,6′′-trideoxy-1-kestose (25). As with brominated compound 24,

chlorinated compound 25 showed no substitution at the 1′′-position. The presence of Br and

Cl isotopic peaks in ESI-MS of compounds 24 and 25 indicated tri-halogenated proportion of

1-kestose, respectively, which supported our analysis.

41

Figure 17 Selected COSY and TOCSY spectrum (500 MHz, CDCl3) of (a)

1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-tribromo-6,6′,6′′-trideoxy-1′-kestose (24); (b)

1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-trichloro-6,6′,6′′-trideoxy-1-kestose (25).

COSY

TOCSY

H-5/H-6b

H-5/H-6a

H-5′/H-6′

H-5′′/H-6′′

H-4/H-5

H-4′/H-5′

H-4′′/H-5′′ H-6a/H-5

H-6b/H-5

H-6′′/H-5′′

H-6′/H-5′

H-6b/H-4

H-6′′/H-4′′

H-6′/H-4′

H-5/H-4

H-5′/H-4′

H-5′′/H-4′′

COSY

TOCSY

H-5/H-6a

H-5′/H-6′

H-5′′/H-6′′

H-4/H-

5H-4′/H-5′

H-4′′/H-5′′ H-6a/H-5

H-6b/H-5

H-6′′/H-5′′

H-6′/H-5′

H-6b/H-4

H-6′′/H-4′′

H-6′/H-4′

H-5/H-4

H-5′/H-4′

H-5′′/H-

4′′

H-5/H-6b

(a)

H-6a H-6b

H-6′′

H-6′H-1′a H-1′b

H-6′′

H-6′H-6a H-6b

H-1′

(b)

42

Based on our result, halogenations of 1-kestose 22 by 4.7 equiv. carbon tetrahalide

(bromide or chloride) and 9.1 equiv. triphenylphosphine at 70 oC for two hours, the only

primary alcohols of 1-kestose that can be contributed to halogenation was at 6-, 6′-, and/or 6′′-

position. As mentioned in the previous paragraph, 1′′-OH of 1′-kestose is considered as the

most hindered position due to its location near the anomeric carbon (or so-called a neopentyl-

like51,75

terminal). These results are in contradiction with chlorination of melezitose (α-D-

glucopyranosyl-(1→3)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, 26) by Appel

reaction (followed by acetylation after first column chromatography) in the previous study.21

By the used of 4 equiv. carbon tetrachloride excess and 8 equiv. triphenylphosphine at 70 oC

for two hours (Scheme 9), 6,1′,6′,6′′-tetrachloro-6,1′,6′,6′′-tetradeoxymelezitose heptaacetate

(27) was isolated in less yield compared with preponderant 6,6′,6′′-trichloro-6,6′,6′′-trideoxy-

melezitose heptaacetate (28). The halogenated proportion at 1′-position of fructofuranosyl

moiety might be formed due to the longer reaction time used in this report, thus unselectively

halogenations occurred at 1′-position. However, no further details of reactivity in Appel

reaction was described in this report (report written in Chinese).

Scheme 9 Previously reported chlorination of melezitose by Appel reactions (27 and 28).21

As for sucrose (1, main skeleton of 1-kestose), the only differentiation between the

primary and secondary OH groups is relatively straightforward: the three primary ones (6-OH,

1′-OH and 6′-OH) are preferentially alkylated, acylated, oxidized or displaced by halogen—an

over-generalization as this order of reactivity mainly covers comparatively bulky reagents

which necessarily favor reaction at the 6-OH and 6′-OH groups.77

Moreover, intra-molecular

hydrogen bonds between 6′-OH and the pyranosidic oxygen atom of the glucose moiety, and

between 1′-OH and O-2 that present in solid state, affected conformation of sucrose (Figure

18). In solution, only one of these bonds remains, involving O-2 and either 1′-OH or 3′-OH.2

Therefore, it is already understood that on the bases of sucrose conformation, 1′-OH is the

most hindered position. Accordingly, the highly selective displacement of the 6-OH and 6′-

OH possibly preferred. However, this explanation is in line with our subjection of 1-kestose.

43

Direct substitution of 1-kestose 22 with 4.7 equiv. carbon tetrahalide (bromide or chloride)

and 9.1 equiv. triphenylphosphine at 70 oC for two hours (Scheme 8) produces pre-O-

acetylated form of halogenated 1-kestose derivatives only at 6,6′,6′′-position (bromination 24

and chlorination 25) .

Figure 18 Sucrose (1) conformational equilibrium.2

B. Deacetylation of per-O-acetylated trihalogenated 1-kestose

To synthesize and elucidate the novel 6,6′,6′′-trihalogenated 1-kestose derivatives,

deacetylation was then conducted. Since suitable deacetylation conditions are generated using

saturated ammonium in methanol for sucrose,75

the undecaacetate of 1-kestose (3) was first

tested for deacetylation and produced 1-kestose (22) within a satisfactory yield (Table 21 in

experiment section). Deacetylation using other methods, such as sodium methoxide, cannot be

used for 1-kestose halogenated derivatives. The per-O-acetylated trihalogenated derivatives of

24 and 25 were underwent deacetylation, which resulted in compound 29 and 30 (Scheme 10).

Scheme 10 Deacetylation of per-O-acetylated 1-kestose derivatives (23–25) to result in 1-

kestose (22) and deacetylated trihalogenated 1-kestose derivatives (29 and 30)

It was complicated to distinguish the halogenated proportion from the deacetylated

compound 29 and/or 30, since the halogenated methylene spin system at H-6, H-6′, and H-6′′

overlapped with other signals such as H-3, H-1′, and/or H-1′′ in the 1H-NMR spectrum. Hence,

the HMBC, HMQC, and HETCOR analyses, together with supports by COSY, TOCSY, and

NOESY, are needed to elucidate the complete structure of deacetylated compounds 29 and 30

(Figure 19). However, structures and halogenation sites of all the deacetylated trihalogenated

44

1-kestose derivatives (29 and 30) were consistent with those in the corresponding per-O-

acetylated forms (24 and 25).

Figure 19 Selected 1H-NMR (500 MHz, D2O) of 1-kestose (22), deacetylated tribrominated

1-kestose derivatives (29), deacetylated trichloronated 1-kestose derivatives (30)

2.3 Experimental section

All reagents used were of analytical grade. NMR spectra were obtained in CDCl3 or

D2O by JEOL EX270 (270 and 67.5 MHz) and JEOL ECA500 (500 and 125 MHz)

spectrometer (JEOL, Tokyo, Japan). Optical rotations were measured at 23 oC on a JASCO

DIP370 polarimeter (JASCO, Tokyo, Japan). HRMS spectra were obtained with a Waters

UPLC ESI-TOF mass spectrometer (Waters, Milford, CT, USA).

2.3.1 Sucrose re-subjection into Appel reaction

Bromination. The bromination was performed in identical procedure.9

A solution of

sucrose (1; 2.00 g, 5.84 mmol) in pyridine (70 mL) was cooled in an ice bath and treated with

triphenylphosphine (3.68 g, 2.4 equiv., 14.03 mmol). A solution of carbon tetrabromide (2.33

g, 1.2 equiv., 7.01 mmol) in pyridine (7 mL) was added dropwise. The reaction mixture was

heated to 60°C and stirred for 1.5 h. After re-cooling the mixture, acetic anhydride (4.6 mL,

0.049 mol) was added, and the mixture was stirred overnight at room temperature. The

solvent was removed under reduced pressure; the residue was washed with methanol and

H-1′a, H-5, H-6, H-6′, H-6′′

H-3, H-1′b, H-1′′H-5′, H-5′′

H-1′a

H-5, H-5′, H-5′′ H-1′b, H-1′′, H-6b, H-3

H-6′

H-6a, H-6′′

Overlap region

H-5′, H-5′′H-4′, H-4′′, H-5

H-6, H-6′, H-6′′, H-1′aH-1′b, H-1′′, H-3

45

ethyl acetate and dissolved in a small amount of dichloromethane, then subjected to column

chromatography on silica gel by elution with hexane/diethyl ether (1:1, 1:2 and 1:3) to yield:

1′,2,3,3′,4,4′-hexa-O-acetyl-6,6′-dibromo-6,6′-dideoxysucrose10,13,14,59,60

(4; 0.28 g, 7%),

eluted first, colorless oil. [α]D +37.6 (c = 1.0, CHCl3); ref.13

[α]D + 42.2 (c =1.0, CHCl3); ref.10

[α]D +46.8 (c = 1.0, CHCl3); ref.14

[α]D +52.5 (c = 0.4, CHCl3); ref.60

[α]D +51 (CHCl3). 1H

NMR (270 MHz, CDCl3): δ = 5.68 (1H, d, J1,2 = 3.6 Hz, H-1), 5.50–5.37 (3H, m, H-3, H-3′,

H-4′), 5.07 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4), 4.88 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 10.6 Hz, H-2),

4.37–4.28 (2H, m, H-5, H-5′), 4.24 (2H, s, H-1′), 3.65 (2H, d, J5′,6′ = 6.6 Hz, H-6′), 3.52 (1H,

dd, J5,6a = 3.0 Hz, J6a,6b = 11.5 Hz, H-6a), 3.40 (1H, dd, J5,6b = 5.6 Hz, J6a,6b = 11.5 Hz, H-6b),

2.18 (3H, s, CH3), 2.16–2.09 (9H, m, 3 x CH3), 2.07 (3H, s, CH3), 2.02 (3H, s, CH3) ppm. 13

C

NMR (67.5 MHz, CDCl3): δ = 170.0, 169.9, 169.9, 169.7, 169.4 (C=O), 104.3 (C-2′), 90.3

(C-1), 81.0 (C-5′), 77.0 (C-4′), 76.1 (C-3′), 70.5 (C-4), 70.1 (C-2), 69.2 (C-3), 69.1 (C-5), 62.3

(C-1′), 31.5 (C-6′), 31.1 (C-6), 20.6, 20.5, 20.5, 20.4, 20.4, 20.4 (CH3) ppm. HRMS (ESI):

calcd. for C24H32O15NaBr2 741.0006, 742.9985, 744.9965 [M + Na]+; found 741.0015,

742.9984, 744.9982.

1′,2,3,3′,4,4′,6-hepta-O-acetyl-6′-bromo-6′-deoxysucrose9 (2; 0.29 g, 7%), eluted second,

colorless oil. [α]D +39.2 (c = 1.0, CHCl3). 1H NMR (270 MHz, CDCl3): δ = 5.65 (1H, d, J1,2 =

3.6 Hz, H-1), 5.50–5.40 (2H, m, H-3, H-3′), 5.37 (1H, t, J3′,4′ = J4′,5′ = 5.1 Hz, H-4′), 5.06 (1H,

t, J3,4 = J4,5 = 9.7 Hz, H-4), 4.88 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 10.2 Hz, H-2), 4.35–4.24 (2H, m,

H-5, H-5′), 4.24–4.17 (3H, m, H-6a, H-1′), 4.17–4.09 (1H, m, H-6b), 3.62 (2H, d, J5′,6′ = 6.6

Hz, H-6′), 2.18 (3H, s, CH3), 2.13–2.10 (12H, m, 4 x CH3), 2.05 (3H, s, CH3), 2.02 (3H, s,

CH3) ppm. 13

C NMR (67.5 MHz, CDCl3): δ = 170.5, 170.0, 169.9, 169.8, 169.7, 169.4 (C=O),

104.3 (C-2′), 90.3 (C-1), 81.1 (C-5′), 77.1 (C-4′), 76.0 (C-3′), 70.1 (C-2), 69.3 (C-3), 68.5 (C-

5), 68.1 (C-4), 62.2 (C-1′), 62.0 (C-6), 31.3 (C-6′), 20.6, 20.6, 20.5, 20.4, 20.4, 20.4,(CH3)

ppm. HRMS (ESI): calcd. for C26H35O17NaBr 721.0955, 723.0935 [M + Na]+; found

721.0959, 723.0907.

1′,2,3,3′,4,4′,6′-hepta-O-acetyl-6-bromo-6-deoxysucrose14

(3; 0.48 g, 12%), eluted third,

colorless oil. [α]D +56.4 (c = 1.0, CHCl3); ref.14

[α]D +49.7 (c = 1.1, CHCl3). 1H NMR (270

MHz, CDCl3): δ = 5.74 (1H, d, J1,2 = 3.6 Hz, H-1), 5.49–5.40 (3H, m, H-3, H-3′, H-4′), 5.11

(1H, t, J3,4 = J4,5 = 9.6 Hz, H-4), 4.87 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 10.6 Hz, H-2), 4.39–4.25

(H-5, m, 3H, H-6′), 4.25–4.21 (1H, m, H-5′), 4.20 (2H, s, H-1′), 3.60 (1H, dd, J5,6a= 3.0 Hz,

J6a,6b= 11.5 Hz, H-6a), 3.42 (1H, dd, J5,6b= 4.6 Hz, J6a,6b= 11.5 Hz, H-6b), 2.17 (3H, s, CH3),

2.12–2.10 (12H, m, 4 x CH3), 2.07 (3H, s, CH3), 2.02 (3H, s, CH3) ppm. 13

C NMR (67.5

MHz, CDCl3): δ = 170.4, 170.0, 169.9, 169.8, 169.6, 169.2 (C=O), 103.9 (C-2′), 89.8 (C-1),

79.0 (C-5′), 75.7 (C-3′), 74.8 (C-4′), 70.6 (C-4), 70.1 (C-2), 69.4 (C-3), 68.8 (C-5), 63.3 (C-

46

6′), 63.0 (C-1′), 31.1 (C-6), 20.6, 20.6, 20.6, 20.5, 20.5 (CH3) ppm. HRMS (ESI): calcd. for

C26H35O17NaBr 721.0955, 723.0935 [M + Na]+; found 721.0946, 723.0933.

Chlorination. Chlorination was similar to bromination as described above, except

carbon tetrabromide in pyridine that was replaced with carbon tetrachloride (0.68 mL, 1.2

equiv., 7.01 mmol) to yield:

1′,2,3,3′,4,4′-hexa-O-acetyl-6,6′-dichloro-6,6′-deoxysucrose4

(8; 0.18 g, 5%), eluted first,

colorless oil. [α]D +49.6 (c = 1.0, CHCl3); ref.4 [α]D +55 (c = 0.55, CHCl3); ref.

13 [α]D +55.8

(c = 1.4, CHCl3); ref.11

[α]D +55.7 (c = 0.3, CHCl3); ref.18

[α]D +49.6 (c = 0.17, CHCl3). 1H

NMR (270 MHz, CDCl3): δ = 5.68 (1H, d, J1,2 = 3.6 Hz, H-1), 5.50–5.35 (3H, m, H-3, H-3′,

H-4′), 5.10 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4), 4.87 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 10.2 Hz, H-2),

4.40–4.31 (1H, m, H-5), 4.29–4.24 (1H, m, H-5′), 4.23 (2H, s, H-1′), 3.79 (2H, d, J5′,6′ = 6.3

Hz, H-6′), 3.67 (1H, dd, J5,6a = 3.0 Hz, J6a,6b = 12.2 Hz, H-6a), 3.56 (1H, dd, J5,6b = 4.9 Hz,

J6a,6b = 12.2 Hz, H-6b), 2.18 (3H, s, CH3), 2.16–2.08 (9H, m, 3 x CH3), 2.07 (3H, s, CH3),

2.02 (3H, s, CH3) ppm. 13

C NMR (67.5 MHz, CDCl3): δ = 170.0, 170.0, 169.7, 169.5, 169.3

(C=O), 104.4 (C-2′), 90.3 (C-1), 81.3 (C-5′), 76.4 (C-4′), 75.9 (C-3′), 70.1 (C-2), 69.6 (C-5),

69.5 (C-4), 69.4 (C-3), 62.4 (C-1′), 44.0 (C-6′), 43.2 (C-6), 20.7, 20.6, 20.5, 20.5, 20.4 (CH3)

ppm. HRMS (ESI): calcd. for C24H32O15NaCl2 653.1016, 655.0986, 657.0957 [M + Na]+;

653.1030, 655.0972, 657.0950.

1′,2,3,3′,4,4′,6-hepta-O-acetyl-6′-chloro-6′-deoxysucrose5,11

(6; 0.33 g, 8%), eluted second,

colorless oil. [α]D +45.2 (c = 1.0, CHCl3); ref.5 [α]D +50 (c = 1.0, CHCl3); ref.

11 [α]D +54 (c =

0.4, CHCl3). 1H NMR (270 MHz, CDCl3): δ = 5.64 (1H, d, J1,2 = 3.6 Hz, H-1), 5.49–5.40 (H-

3, m, 2H, H-3′), 5.37 (1H, t, J3′,4′ = J4′,5′ = 5.1 Hz, H-4′), 5.06 (1H, t, J3,4 = J4,5 = 9.9 Hz, H-4),

4.87 (1H, dd, J1,2 = 3.6 Hz, J2,3 = 10.2 Hz, H-2), 4.33–4.25 (1H, m, H-5), 4.25–4.17 (4H, m,

H-6a, H-1′, H-5′), 4.13 (1H, dd, J5,6b = 2.0 Hz, J6a,6b = 12.2 Hz, H-6b) 3.77 (2H, d, J5′,6′ = 6.6

Hz, H-6′), 2.18 (3H, s, CH3), 2.15–2.08 (12H, m, 4 x CH3), 2.05 (3H, s, CH3), 2.02 (3H,s,

CH3) ppm. 13

C NMR (67.5 MHz, CDCl3): δ = 170.6, 170.0, 169.9, 169.8, 169.5 (C=O), 104.4

(C-2′), 90.3 (C-1), 81.3 (C-5′), 76.5 (C-4′), 76.0 (C-3′), 70.2 (C-2), 69.4 (C-3), 68.5 (C-5),

68.2 (C-4), 62.4 (C-1′), 62.0 (C-6), 43.9 (C-6′), 20.7, 20.6, 20.6, 20.5, 20.4 (CH3) ppm.

HRMS (ESI): calcd. for C26H35O17NaCl 677.1460, 679.1431 [M + Na]+; found 677.1463,

679.1426.

1′,2,3,3′,4,4′,6′-hepta-O-acetyl-6-chloro-6-deoxysucrose5,14

(7; 0.51 g, 13%), eluted third,

colorless oil. [α]D +60.0 (c = 1.0, CHCl3); ref.5 [α]D +69 (c = 1.0, CHCl3); ref.

14 [α]D +52.7 (c

= 0.45, CHCl3). 1

H NMR (270 MHz, CDCl3): δ = 5.74 (1H, d, J1,2 = 3.6 Hz, H-1), 5.49–5.39

(H-3, m, 3H, H-3′, H-4′), 5.14 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4), 4.87 (1H, dd, J1,2 = 3.6 Hz, J2,3

= 10.6 Hz, H-2), 4.41–4.26 (3H, m, H-5, H-6′), 4.25–4.21 (1H, m, H-5′), 4.20 (2H, s, H-1′),

47

3.73 (1H, dd, J5,6a = 3.0 Hz, J6a,6b = 12.2 Hz, H-6a), 3.58 (1H, dd, J5,6b = 4.6 Hz, J6a,6b = 12.2

Hz, H-6b), 2.17 (3H, s, CH3), 2.13–2.08 (12H, m, 4 x CH3), 2.06 (3H, s, CH3), 2.02 (3H,s,

CH3) ppm. 13

C NMR (67.5 MHz, CDCl3): δ = 170.2, 169.8, 169.7, 169.6, 169.4, 169.1 (C=O),

103.7 (C-2′), 89.6 (C-1), 78.8 (C-5′), 75.5 (C-3′), 74.6 (C-4′), 70.0 (C-2), 69.4 (C-3), 69.2 (C-

4), 69.2 (C-5), 63.1 (C-6′), 62.8 (C-1′), 43.0 (C-6), 20.4, 20.4, 20.4 (CH3) ppm. HRMS (ESI):

calcd. for C26H35O17NaCl 677.1460, 679.1431 [M + Na]+; found 677.1464, 679.1449.

2.3.2 Chemoenzymatic Synthesis of 1′-Halodeoxysucrose Derivatives

2,3,3′,4,4′,6,6′-Hepta-O-acetyl-sucrose (9): The procedure was described in ref.65–67

A

solution of sucrose octaacetate (5; 1.00 g, 1.47 mmol) in 0.1 M sodium phosphate (pH 7.0)–

DMF (3:1) was treated with Alcalase 2.4 L (2.50 g) and then incubated at 37°C for 24 h. The

reaction was quenched by extracting the products with ethyl acetate. The extract was

concentrated under reduced pressure and the residue was subjected to column

chromatography on silica gel by elution with EtOAc/hexane (20:1 and 5:1) to yield 9 (0.25 g,

27%).

2,3,3′,4,4′,6,6′-hepta-O-acetyl-1′-bromo-1′-deoxysucrose (10): The procedure was described

in ref.6 A solution of pyridine (0.59 mL) and Tf2O (0.61 mL) in CH2Cl2 (10 mL) was added

dropwise to a solution of 9 (0.50 g, 0.78 mmol) in CH2Cl2 (10 mL) at 0°C. After 20 min, the

mixture was poured into a saturated NaHCO3 solution and partitioned with CH2Cl2. The

organic layer was washed with 1 M HCl and saturated NaHCO3, and then dried over MgSO4.

The solvent was removed under reduced pressure. The residue was dissolved in DMF (10 mL)

and LiBr (0.27 g, 4.0 equiv., 3.12 mmol) was added to the solution. After 2.5 h, the mixture

was diluted with EtOAc (30 mL) and extracted with water (20 mL). The organic layer was

washed with brine, dried over MgSO4, and evaporated. The residue was subjected to column

chromatography on silica gel by elution with hexane–EtOAc (2:1) to yield 10 (0.46 g, 85%)

as colorless solid. [α]D +52.6 (c = 1.0, CH3Cl3). 1H NMR (500 MHz, CDCl3): δ = 5.74–5.71

(2H, m, H-1, H-3′), 5.47–5.40 (2H, m, H-3, H-4′), 5.08 (1H, t, J4,5 = 9.5 Hz, H-4), 4.91 (1H,

dd, J1,2 = 4.6 Hz, J2,3 = 10.3 Hz, H-2), 4.34–4.26 (4H, m, H-5, H-6a, H-6′), 4.24–4.20 (1H, m,

H-5′), 4.16 (1H, d, J6a,6b = 11.5 Hz, H-6b), 3.61 (1H, d, J1′a,1′b = 12.0 Hz, H-1′a), 3.49 (1H, d,

J1′a,1′b = 12.0 Hz, H-1′b), 2.17 (3H, s, CH3), 2.12 (3H, s, CH3), 2.11–2.09 (6H, m, 2 x CH3),

2.08 (3H, s, CH3), 2.04 (3H,s, CH3), 2.02 (3H, s, CH3) ppm. 13

C NMR (125 MHz, CDCl3): δ =

170.6, 170.4, 170.0, 169.9, 169.7, 169.6, 169.4 (C=O), 103.4 (C-2′), 89.7(C-1), 78.9 (C-5′),

76.1 (C-3′), 74.0 (C-4′), 69.9 (C-2), 69.6 (C-3), 68.4 (C-5), 68.0 (C-4), 63.1 (C-6′), 61.6 (C-6),

33.5 (C-1′), 20.7, 20.6, 20.6, 20.5, 20.5 (CH3) ppm. HRMS (ESI): calcd. for C26H35O17NaBr

721.0955, 723.0935 [M + Na]+; found 721.0959, 723.0931.

48

2,3,3′,4,4′,6,6′-hepta-O-acetyl-1′-chloro-1′-deoxysucrose8,68

(11): The procedure was

described in ref.6 The procedure was similar to the synthesis of 10 as described above, except

LiBr was replaced with LiCl (0.13 g, 4.0 equiv., 3.12 mmol) to yield 11 (0.37 g, 72%) as

colorless solid. [α]D +46.4 (c = 1.0, CH3Cl3); ref.8 [α]D +53.8 (c = 2.68, CH3Cl3); ref.

68 [α]D

+55.0 (c = 1.2, CH3Cl3). 1

H NMR (500 MHz, CDCl3): δ = 5.72 (1H, d, J1,2 = 4.0 Hz, H-1),

5.69 (1H, d, J3′,4′ = 6.3 Hz, H-3′), 5.46–5.41 (2H, m, H-3, H-4′), 5.08 (1H, t, J4,5 = 9.7 Hz, H-

4), 4.91 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 10.3 Hz, H-2), 4.36–4.27 (4H, m, H-5, H-6a, H-6′), 4.22

(1H, td, J4′,5′ = 6.4 Hz, J5′,6′ = 3.6 Hz, H-5′), 4.16 (1H, dd, J5,6b = 2.9 Hz, J6a,6b = 13.2 Hz, H-

6b), 3.74 (1H, d, J1′a,1′b = 12.0 Hz, H-1′a), 3.57 (1H, d, J1′a,1′b = 12.0 Hz, H-1′b), 2.18 (3H, s,

CH3), 2.13 (3H, s, CH3), 2.10 (6H, s, 2 x CH3), 2.07 (3H, s, CH3), 2.04 (3H, s, CH3), 2.02

(3H, s, CH3) ppm. 13

C NMR (125 MHz, CDCl3): δ = 170.6, 170.4, 170.0, 169.9, 169.8, 169.6,

169.4 (C=O), 103.9 (C-2′), 89.5 (C-1), 78.9 (C-5′), 75.4 (C-3′), 73.9 (C-4′), 69.9 (C-2), 69.6

(C-3), 68.4 (C-5), 68.0 (C-4), 63.0 (C-6′), 61.5 (C-6), 45.3 (C-1′), 20.6, 20.6, 20.6, 20.5 (CH3)

ppm. HRMS (ESI): calcd. for C26H35O17NaCl 677.1460, 679.1431 [M + Na]+; found

677.1459, 679.1436.

2.3.3 1-Kestose subjection into Appel Reaction

Bromination. Solution of 1-kestose (22, 0.10 g, 0.198 mmol) in pyridine (2 mL) was

cooled in an ice bath and treated with triphenylphosphine (0.47 g, 1.8 mmol, 9.1 equiv),

followed by dropwise addition of a solution of carbon tetrabromide (0.30 g, 0.93 mmol, 4.7

equiv) in pyridine (0.93 mL). The reaction mixture was stirred at 70 °C for 2 h. After cooling

the mixture in an ice bath, acetic anhydride (3 mL, 32.0 mmol) and pyridine (2 mL) were

added, and the mixture was stirred overnight at room temperature. The solvent was

evaporated and the residue was partitioned between CH2Cl2 and water. The organic layer was

washed with 1 M HCl and brine, dried over MgSO4, reduced the solvent under high pressure

and then purified by column chromatography on silica gel by elution with hexane/diethyl

ether (1:6 and 1:10) to yield 1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-tribromo-6,6′,6′′-

trideoxy 1-kestose (24; 0.0516 g, 25%) as colorless amorphous mass. [α]D +22.4 (c = 1.0,

CHCl3). 1H-NMR (500 MHz, CDCl3) δ: 5.70 (1H, d, J3′,4′ = 8.0 Hz, H-3′), 5.67 (1H, d, J1,2 =

4.0 Hz, H-1), 5.50 (1H, d, J3′′,4′′ = 6.3 Hz, H-3′′), 5.48–5.42 (2H, m, H-3, H-4′), 5.32 (1H, t,

J3′′,4′′ = 6.3 Hz, H-4′′), 5.03 (1H, t, J3,4 = 9.7 Hz, H-4), 4.95 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 10.3

Hz, H-2), 4.35–4.28 (2H, m, H-5, H-5′), 4.24 (2H, s, H-1′′), 4.22–4.19 (1H, m, H-5′′), 3.79

(1H, d, J1′a,1′b = 10.3 Hz, H-1′a), 3.73 (1H, d, J1′a,1′b = 10.3 Hz, H-1′b), 3.69 (2H, d, J5′′,6′′ = 7.4

Hz, H-6′′), 3.66 (2H, d, J5′,6′ = 6.9 Hz, H-6′), 3.54 (1H, dd, J5,6a = 2.3 Hz, J6a,6b =11.5 Hz, H-

6a), 3.40 (1H, dd, J5,6b = 6.3 Hz, J6a,6b =11.5 Hz, H-6b), 2.16–2.15 (6H, m, 2 x CH3), 2.14 (3H,

49

s, CH3), 2.11 (3H, s, CH3), 2.09–2.07 (9H, m, 3 x CH3), 2.02 (3H, s, CH3) ppm. 13

C-NMR

(125 MHz, CDCl3) δ: 170.2, 170.0, 170.0, 169.8, 169.5 (C=O), 103.8 (C-2′), 103.1 (C-2′′),

90.1 (C-1), 80.7 (C-5′′), 79.8 (C-5′), 78.1 (C-4′′), 77.2 (C-3′′), 76.4 (C-4′), 75.8 (C-3′), 70.8

(C-4), 69.7 (C-2), 69.3 (C-3 & C-5), 62.0 (C-1′′), 61.5 (C-1′), 32.7 (C-6′′), 32.0 (C-6′), 31.1

(C-6), 20.8, 20.8, 20.7, 20.8, 20.6, 20.5, 20.5, 20.5 (CH3) ppm. HRMS (ESI): calcd. for

C34H45O21Br3Na 1050.9881, 1052.9860 [M + Na]+; found 1050.9899, 1052.9880.

Chlorination. Chlorination was similar to bromination as described above, except

carbon tetrabromide in pyridine was replaced with carbon tertrachloride (0.09 mL, 0.93 mmol,

4.7 equiv.) to yield 1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-trichloro-6,6′,6′′-trideoxy-1-

kestose (25; 0.0514 g, 28%) as colorless amorphous mass. [α]D +26.6 (c = 1.0, CHCl3). 1H-

NMR (500 MHz, CDCl3) δ: 5.70–5.66 (2H, m, H-1, H-3′), 5.50 (1H, d, J3′′,4′′ = 6.3 Hz, H-3′′),

5.48–5.41 (2H, m, H-3, H-4′), 5.33 (1H, t, J3′′,4′′ = 6.3 Hz, H-4′′), 5.06 (1H, t, J3,4 = 9.7 Hz, H-

4), 4.93 (1H, dd, J1,2 = 3.7 Hz, J2,3 = 10.6 Hz, H-2), 4.38–4.32 (1H, m, H-5), 4.28–4.22 (3H,

m, H-1′′, H-5′), 4.17 (1H, q, J5′′,6′′ = 6.5 Hz, H-5′′), 3.82–3.78 (4H, m, H-6′, H-6′′), 3.75 (2H, s,

H-1′), 3.68 (1H, dd, J5,6a = 2.3 Hz, J6a,6b = 12.0 Hz, H-6a), 3.57 (1H, dd, J5,6b = 5.7 Hz, J6a,6b =

12.0 Hz, H-6b), 2.16 (3H, s, CH3), 2.15–2.13 (6H, m, 2 x CH3), 2.11 (3H, s, CH3), 2.09 (3H, s

CH3), 2.08 (3H, s, CH3), 2.07 (3H, s, CH3), 2.02 (3H, s, CH3). 13

C-NMR (125 MHz, CDCl3)

δ: 170.2, 170.1, 170.0, 170.0, 169.7, 169.5 (C=O), 103.8 (C-2′), 103.1 (C-2′′), 90.0 (C-1), 80.6

(C-5′′), 79.9 (C-5′), 77.2 (C-4′′), 76.9 (C-3′′), 75.7 C-4′), 75.5 (C-3′), 69.8 (C-5), 69.7 (C-2 &

C-4), 69.4 (C-3), 62.2 (C-1′′), 61.6(C-1′), 44.6 (C-6′′), 44.4 (C-6′), 43.2 (C-6), 20.8, 20.7, 20.7,

20.6, 20.5, 20.5 (CH3) ppm. HRMS (ESI): calcd. for C34H45O21Cl3Na 919.1387 [M + Na]+;

found 919.1395.

2.3.4 Acetylation of Sucralose

4,1′,6′-Trichloro-4,1′,6′-trideoxygalactosucrose78

(12): 1H NMR (500 MHz, D2O): δ = 5.44

(1H, d, J1,2 = 4.0 Hz, H-1), 4.49 (1H, d, J3,4 = J4,5 = 4.0 Hz, H-4), 4.40–4.36 (2H, m, H-3′, H-

5), 4.15 (1H, dd, J2,3 = 10.3 Hz, J3,4 = 4.0 Hz, H-3), 4.09 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′),

4.06–4.02 (1H, m, H-5′), 3.92 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 10.3 Hz, H-2), 3.87 (2H, dd, J5′,6′a

= 5.2 Hz, J5′,6′b = 2.9 Hz, H-6′), 3.76 (2H, brs, H-1′), 3.73 (2H, d, J5,6 = 6.3 Hz, H-6) ppm. 13

C-

NMR (125 MHz, D2O): δ = 103.3 (C-2′), 92.6 (C-1), 81.2 (C-5′), 75.9 (C-3′), 75.2 (C-4′),

70.7 (C-5), 68.0 (C-3), 67.5 (C-2), 62.9 (C-4), 61.3 (C-6), 44.7 (C-6′), 43.4 (C-1′) ppm.

2,3,3′,4′,6-Penta-O-acetyl-4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose5,79

(13):

Compound 12 (0.21 g, 0.53 mmol) was dissolved in pyridine (8 mL) and treated with acetic

anhydride (0.5 mL, 5.30 mmol). The mixture was then stirred overnight at room temperature.

The solvent was removed under reduce pressure and subjected to column chromatography on

50

silica gel by elution with chloroform to yield 13 (0.25 g, 78%) as colorless liquid. [α]D +63.5

(c = 1.0, CHCl3); ref.79

[α]D +66.8 (c = 0.9, CHCl3). 1H NMR (500 MHz, CDCl3): δ = 5.70

(1H, d, J3′,4′ = 6.3 Hz, H-3′), 5.68 (1H, d, J1,2 = 1.7 Hz, H-1), 5.41 (1H, t, J3′,4′ = J4′,5′ = 6.3 Hz,

H-4′), 5.31–5.28 (2H, m, H-2, H-3), 4.60–4.54 (2H, m, H-4, H-5), 4.28–4.21 (3H, m, H-6, H-

5′), 3.77 (2H, d, J5′,6′ = 5.7 Hz, H-6′), 3.71 (1H, d, J1′a,1′b = 12.0 Hz, H-1′a), 3.60 (1H, d, J1′a,1′b

= 12.0 Hz, H-1′b), 2.15 (3H, s, CH3), 2.14 (3H, s, CH3), 2.12 (3H, s, CH3), 2.10 (3H, s, CH3),

2.10 (3H, s, CH3) ppm. 13

C NMR (125 MHz, CDCl3): δ = 170.4, 170.0, 169.8, 169.6 (C=O),

104.3 (C-2′), 90.6 (C-1), 80.7 (C-5′), 76.0 (C-4′), 75.8 (C-3′), 67.9 (C-2), 67.7 (C-5), 66.8 (C-

3), 63.5 (C-6), 58.9 (C-4), 44.4 (C-1′), 43.8 (C-6′), 20.8, 20.7, 20.7, 20.5 (CH3) ppm. HRMS

(ESI): calcd. for C22H29O13NaCl3 629.0571, 631.0542 [M + Na]+; found 629.0574, 631.0544.

2.3.5 Acetylation of 1-kestose

1′′,2,3,3′,3′′,4,4′,4′′,6,6′,6′′-Undeca-O-acetyl-deoxy-1-kestose (23):76,80

Solution of 1-

kestose (22, 0.03 g, 0.06 mmol) in pyridine (15 mL) was treated with acetic anhydride (1 mL,

10.7 mmol), and the mixture was stirred overnight at room temperature. The solvent was

removed under reduced pressure and subjected to column chromatography on silica gel by

elution with chloroform to yield 23 (0.055 g, 96%) as a colorless liquid. [α]D + 30.2 (c = 1.0,

CHCl3); Ref.80

[α]D +31.8 (c = 3.7, CHCl3). 1H-NMR (500 MHz, CDCl3) δ: 5.75 (1H, d, J1,2 =

3.4 Hz, H-1), 5.69 (1H, d, J3′,4′ = 8.0 Hz, H-3′), 5.48 (1H, d, J3′′,4′′ = 6.9 Hz, H-3′′), 5.46 (1H, t,

J3′,4′ = 8.0 Hz, H-4′), 5.42 (1H, t, J3,4 = 9.7 Hz, H-3), 5.34 (1H, t, J3′′,4′′ = 6.9 Hz, H-4′′), 5.08

(1H, t, J3,4 = 9.7 Hz, H-4), 4.91 (1H, dd, J1,2 = 3.4 Hz, J2,3 = 10.3 Hz, H-2), 4.39–4.33 (3H, m,

H-5, H-6′′), 4.33–4.24 (3H, m, H-6a, H-6′), 4.24–4.20 (3H, m, H-1′′, H-5′), 4.20–4.14 (2H, m,

H-6b, H-5′′), 3.69 (1H, d, J1′a,1′b = 9.2 Hz, H-1′a), 3.63 (1H, d, J1′a,1′b = 9.2 Hz, H-1′b), 2.19–

2.14 (3H, m, CH3), 2.13–2.12 (6H, m, 2 x CH3), 2.11–2.09 (15H, m, 5 x CH3), 2.06 (3H, s,

CH3), 2.04 (3H, s, CH3), 2.01 (3H, s, CH3) ppm. 13

C-NMR (125 MHz, CDCl3) δ: 170.7, 170.6,

170.5, 170.1, 169.9, 169.7, 169.6 (C=O), 103.4 (C-2′), 102.9 (C-2′′), 89.2 (C-1), 78.4 (C-5′′),

77.8 (C-5′), 76.5 (C-3′′), 75.5 (C-4′′), 74.9 (C-3′), 73.7 (C-4′), 70.0 (C-2), 69.8 (C-3), 68.2 (C-

5), 68.2 (C-4), 63.7 (C-6′′), 63.2 (C-6′), 62.7 (C-1′′), 62.2 (C-1′), 61.7(C-6), 20.8, 20.7, 20.7,

20.6, 20.6, 20.5 (CH3) ppm. HRMS (ESI): calcd. for C40H54O27Na 989.2750 [M + Na]+; found

989.2797.

2.3.6 Deacetylation of per-O-acetylated halogenated carbohydrates

A. Deacetylation of per-O-acetylated Halodeoxysucrose Derivatives

General procedure for deacetylation of per-O-acetylated halodeoxysucrose

derivatives: Solutions of per-O-acetylated halodeoxysucrose derivatives 2–4, 6–8,

51

10 and 11 in dry methanol were treated with saturated ammonia in methanol and

stirred overnight at room temperature. The solvent was removed under reduce

pressure and subjected to column chromatography on silica gel (pre-washed by

methanol) by elution with methanol/chloroform. The product then was washed by

diethyl ether, ethyl acetate, acetonitrile, and water—chloroform (1:1). The method

afforded the corresponding halodeoxysucrose derivatives 14–21.

6′-Bromo-6′-deoxysucrose (14; 48 mg, 98%): Colorless amorphous mass. [α]D +64.6 (c = 1.0,

CH3OH). 1H NMR (500 MHz, D2O): δ = 5.34 (1H, d, J1,2 = 4.0 Hz, H-1), 4.17 (1H, d, J3′,4′ =

8.0 Hz, H-3′), 4.04 (1H, t, J3′,4′ = J4′,5′ = 8.0 Hz, H-4′), 4.02–3.98 (1H, m, H-5′), 3.84–3.78

(2H, m, H-5, H-6a), 3.73–3.66 (4H, m, H-3, H-6b, H-6′), 3.64 (2H, brs, H-1′), 3.50 (1H, dd,

J1,2= 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.36 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4) ppm. 13

C NMR (125

MHz, D2O): δ = 103.9 (C-2′), 92.4 (C-1), 80.8 (C-5′), 77.0 (C-4′), 76.6 (C-3′), 72.7 (C-5),

72.6 (C-3), 71.1 (C-2), 69.5 (C-4), 61.0 (C-1′), 60.5 (C-6), 33.2 (C-6′) ppm. HRMS (ESI):

calcd. for C12H21O10NaBr 427.0216, 429.0195 [M + Na]+; found 427.0213, 429.0208.

6-Bromo-6-deoxysucrose14

(15; 75 mg, 90%): Colorless amorphous mass. [α]D +43.6 (c = 1.0,

CH3OH). 1H NMR (500 MHz, D2O): δ = 5.38 (1H, d, J1,2 = 4.0 Hz, H-1), 4.18 (1H, d, J3′,4′ =

8.6 Hz, H-3′), 4.05 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 3.99 (1H, dt, J4,5 = 9.7 Hz, J5,6 = 3.4 Hz,

H-5), 3.86 (1H, td, J4′,5′ = 8.6 Hz, J5′,6′ = 3.4 Hz, H-5′), 3.81–3.76 (2H, m, H-6′), 3.76–3.71

(3H, m, H-3, H-6), 3.63 (2H, brs, H-1′), 3.55 (1H, dd, J1,2= 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.47

(1H, t, J3,4 = J4,5 = 9.7 Hz, H-4) ppm. 13

C NMR (125 MHz, D2O): δ = 103.7 (C-2′), 92.1(C-1),

81.3 (C-5′), 76.2 (C-3′), 74.0 (C-4′), 72.1 (C-3), 71.2 (C-4), 71.0 (C-2), 70.7 (C-5), 62.5 (C-

6′), 61.4 (C-1′), 33.4 (C-6) ppm. HRMS (ESI): calcd. for C12H21O10NaBr 427.0216, 429.0195

[M + Na]+; found 427.0231, 429.0208.

6,6′-Dibromo-6,6′-dideoxysucrose10

(16; 94 mg, 80%): Colorless amorphous mass. [α]D

+37.4 (c = 1.0, CH3OH); ref.10

[α]D +27.4 (c = 1.0, CH3OH); ref.60

[α]D +37 (c = 1.0,

CH3OH). 1H NMR (500 MHz, D2O): δ = 5.35 (1H, d, J1,2 = 4.0 Hz, H-1), 4.17 (1H, d, J3′,4′ =

8.6 Hz, H-3′), 4.08 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 4.01–4.00 (2H, m, H-5, H-5′), 3.75–

3.69 (3H, m, H-6′, H-3), 3.69–3.65 (1H, m, H-6a), 3.64 (2H, brs, H-1′), 3.63–3.60 (1H, m, H-

6b), 3.52 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.42 (1H, t, J3,4 = J4,5 = 9.5 Hz, H-4) ppm.

13C NMR (125 MHz, D2O): δ = 103.8 (C-2′), 92.3 (C-1), 80.5 (C-5′), 76.6 (C-4′), 76.4 (C-3′),

72.0 (C-3), 71.4 (C-4), 71.0 (C-5), 71.0 (C-2), 61.1 (C-1′), 33.3 (C-6), 33.2 (C-6′) ppm.

HRMS (ESI): calcd. for C12H20O9NaBr2 488.9372, 490.9351, 492.9331 [M + Na]+; found

488.9354, 490.9356, 492.9329.

52

6′-Chloro-6′-deoxysucrose81

(17; 71 mg, 82%): Colorless amorphous mass. [α]D +59.0 (c =

1.0, CH3OH); ref.5 [α]D +64 (c = 1.0, H2O).

1H NMR (500 MHz, D2O): δ = 5.33 (1H, d, J1,2 =

3.4 Hz, H-1), 4.17 (1H, d, J3′,4′ = 8.6 Hz, H-3′), 4.06 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 3.97

(1H, dd, J4′,5′ = 8.6 Hz, J5′,6′ = 5.7 Hz, H-5′), 3.85–3.77 (4H, m, H-6a, H-6′, H-5), 3.73–3.66

(2H, m, H-3, H-6b), 3.64 (2H, brs, H-1′), 3.49 (1H, dd, J1,2 = 3.4 Hz, J2,3 = 9.7 Hz, H-2), 3.36

(1H, t, J3,4 = J4,5 = 9.7 Hz, H-4) ppm. 13

C NMR (125 MHz, D2O): δ = 103.8 (C-2′), 92.3 (C-

1), 80.7 (C-5′), 76.3 (C-3′), 75.9 (C-4′), 72.6 (C-5), 72.5 (C-3), 71.0 (C-2), 69.4 (C-4), 60.9

(C-1′), 60.3 (C-6), 45.1 (C-6′) ppm. HRMS (ESI): calcd. for C12H21O10NaCl 383.0721,

385.0691 [M + Na]+; found 383.0721, 385.0704.

6-Chloro-6-deoxysucrose4,5

(18; 55 mg, 83%): Colorless amorphous mass. [α]D +39.2 (c =

1.0, CH3OH); ref.4 [α]D +55 (c = 1, H2O); ref.

5 [α]D +46 (c = 1.0, H2O).

1H NMR (500 MHz,

D2O): δ = 5.36 (1H, d, J1,2 = 3.4 Hz, H-1), 4.16 (1H, d, J3′,4′ = 8.6 Hz, H-3′), 4.05 (1H, dt, J4,5

= 9.7 Hz, J5,6 = 3.2 Hz, H-5), 4.00 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 3.86–3.82 (3H, m, H-6,

H-5′), 3.77 (2H, d, J5′,6′ = 2.9 Hz, H-6′), 3.72–3.69 (1H, m, H-3), 3.61 (2H, brs, H-1′), 3.52

(1H, dd, J1,2 = 3.4 Hz, J2,3 = 9.7 Hz, H-2), 3.47 (1H, t, J3,4 = J4,5 = 9.7 Hz, 4H) ppm. 13

C NMR

(125 MHz, D2O): δ = 103.7 (C-2′), 92.1 (C-1), 81.3 (C-5′), 76.1 (C-3′), 74.0 (C-4′), 72.1 (C-

3), 71.3 (C-5), 71.0 (C-2), 69.9 (C-4), 62.5 (C-6′), 61.3 (C-1′), 44.2 (C-6) ppm. HRMS (ESI):

calcd. for C12H21O10NaCl 383.0721, 385.0691 [M + Na]+; found 383.0721, 385.0695.

6,6′-Dichloro-6,6′-dideoxysucrose10,69

(19; 59 mg, 94%): Colorless amorphous mass. [α]D

+43.6 (c = 1.0, CH3OH); ref.4 [α]D +60 (c = 0.5, H2O); ref.

10 [α]D +50.4 (c = 0.9, CH3OH);

ref.58

[α]D +58 (c = 0.5, H2O); ref.15

[α]D +60 (c = 1, H2O). 1

H NMR (500 MHz, D2O): δ =

5.39 (1H, d, J1,2 = 4.0 Hz, H-1), 4.21 (1H, d, J3′,4′ = 8.6 Hz, H-3′), 4.14–4.08 (2H, m, H-5, H-

4′), 4.00 (1H, td, J 4′,5′ = 8.0 Hz, J5′,6′= 3.4 Hz, H-5′), 3.91–3.84 (2H, m, H-6), 3.84–3.78 (2H,

m, H-6′), 3.74 (1H, t, J2,3 = J3,4 = 9.7 Hz, H-3), 3.67 (2H, brs, H-1′), 3.55 (1H, dd, J1,2 = 4.0

Hz, J2,3 = 9.7 Hz, H-2), 3.48 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-4) ppm. 13

C NMR (125 MHz,

D2O): δ = 103.9 (C-2′), 92.3 (C-1), 80.7 (C-5′), 76.3 (C-3′), 75.6 (C-4′), 72.1 (C-3), 71.6 (C-

5), 71.0 (C-2), 70.1 (C-4), 61.2 (C-1′), 45.2 (C-6′), 44.3 (C-6) ppm. HRMS (ESI): calcd. for

C12H20O9NaCl2 401.0382, 403.0353, 405.0323 [M + Na]+; found 401.0400, 403.0364,

405.0331.

1′-Bromo-1′-deoxysucrose (20; 8.7 mg, 39%): Colorless amorphous mass. [α]D +36.4 (c =

1.0, CH3OH). 1H NMR (500 MHz, D2O): δ = 5.40 (1H, d, J1,2 = 4.0 Hz, H-1), 4.42 (1H, d,

J3′,4′ = 8.6 Hz, H-3′), 4.01 (1H, t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 3.87 (1H, td, J4′,5′ = 8.6 Hz, J5′,6′ =

2.9 Hz, H-5′), 3.82–3.76 (5H, m, H-5, H-6, H-6′), 3.73–3.69 (2H, m, H-3, H-1′a), 3.63 (1H,

brs, H-1′b), 3.52 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.44 (1H, t, J3,4 = J4,5 = 9.7 Hz, H-

4) ppm. 13

C NMR (125 MHz, D2O): δ = 102.8 (C-2′), 92.7 (C-1), 81.9 (C-5′), 77.0 (C-3′),

53

73.6 (C-4′), 72.7 (C-3 & C-5), 71.1 (C-2), 69.4 (C-4), 62.1(C-6′), 60.2 (C-6), 32.6 (C-1′) ppm.

HRMS (ESI): calcd. for C12H21O10NaBr 427.0216, 429.0195 [M + Na]+; found 427.0212,

429.0191.

1′-Chloro-1′-deoxysucrose8,82

(21; 2.8 mg, 21%): Colorless amorphous mass. [α]D +40.0 (c =

1.0, CH3OH); ref.8 [α]D +60.4 (c = 1.0, H2O); ref.

68 [α]D +57.8 (c = 0.7, H2O).

1H NMR (500

MHz, D2O): δ = 5.40 (1H, d, J1,2 = 4.0 Hz, H-1), 4.36 (1H, d, J3′,4′ = 8.6 Hz, H-3′), 4.01 (1H,

t, J3′,4′ = J4′,5′ = 8.6 Hz, H-4′), 3.88 (1H, td, J4′,5′ = 8.6 Hz, J5′,6′ = 3.4 Hz, H-5′), 3.81 (1H, dd,

J5,6a = 2.9 Hz, J5,6b = 5.7 Hz, H-5), 3.80–3.77 (4H, m, H-6, H-6′), 3.77 (2H, brs, H-1′), 3.71

(1H, t, J3,4 = 9.7 Hz, H-3), 3.52 (1H, dd, J1,2= 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.43 (1H, t, J3,4 = J4,5

= 9.7 Hz, H-4) ppm. 13

C NMR (125 MHz, D2O): δ = 103.1 (C-2′), 92.5 (C-1), 81.7 (C-5′),

76.2 (C-3′), 73.4 (C-4′), 72.5 (C-3 & C-5), 70.9 (C-2), 69.2 (C-4), 61.9 (C-6′), 60.0 (C-6),

43.8 (C-1′) ppm. HRMS (ESI): calcd. for C12H21O10NaCl 383.0721, 385.0691 [M + Na]+;

found 383.0723, 385.0695.

B. Deacetylation of per-O-acetylated halodeoxy-1-kestose derivative

Solutions of per-O-acetylated halodeoxy-1-kestose derivatives (23–25, 0.05 g) in 3 mL

dry methanol were treated with 1.5 mL saturated ammonia in methanol and stirred overnight

at room temperature. The solvent was removed under reduced pressure and subjected to

column chromatography on silica gel (pre-washed by methanol) by elution with

methanol/chloroform (3:1). The method produced the corresponding halodeoxysucrose

derivatives 22, 29, and 30.

1-kestose71

(22, 0.034 g, 97%). 1H-NMR (500 MHz, D2O) δ: 5.37 (1H, d, J1,2 = 4.0 Hz,

H-1), 4.22 (1H, d, J3′,4′ = 8.6 Hz, H-3′), 4.13 (1H, d, J3′′,4′′ = 8.6 Hz, H-3′′), 4.02 (1H, t, J3′′,4′′ =

8.6 Hz, H-4′′), 3.98 (1H, t, J3′,4′ = 8.6 Hz, H-4′), 3.83–3.80 (2H, m, H-5′, H-5′′), 3.79–3.71 (8H,

m, H-1′a, H-5, H-6, H-6′, H-6′′), 3.70–3.60 (4H, m, H-3, H-1′b, H-1′′), 3.48 (1H, dd, J1,2 = 4.0

Hz, J2,3 = 9.7, H-2), 3.41 (1H, t, J3,4 = 9.7 Hz, H-4) ppm. 13

C-NMR (125 MHz, D2O) δ: 104.2

(C-2′′), 103.7 (C-2′), 92.9 (C-1), 81.6 (C-5′), 81.5 (C-5′′), 77.0 (C-3′ & C-3′′), 74.9 (C-4′′),

74.2 (C-4′), 73.0 (C-3), 72.8 (C-5), 71.6 (C-2), 69.6 (C-4), 62.7 (C-6′′), 62.6 (C-6′), 61.3 (C-

1′), 60.8 (C-1′′), 60.5 (C-6) ppm.

6,6′,6′′-tribromo-6,6′,6′′-trideoxy-1-kestose (29, 0.030 g, 81%). [α]D = + 24.8 (c 1.0,

MeOH). 1H-NMR (500 MHz, D2O) δ: 5.36 (1H, d, J1,2 = 4.0 Hz, H-1), 4.27 (1H, d, J3′,4′ = 8.6

Hz, H-3′), 4.16 (1H, d, J3′′,4′′ = 8.6 Hz, H-3′′), 4.09–4.05 (2H, m, H-4′, H-4′′), 4.04–3.98 (3H,

m, H-5, H-5′, H-5′′), 3.87 (1H, d, J1′a,1′b = 10.3 Hz, H-1′a), 3.79–3.72 (3H, m, H-6a, H-6′′),

3.72–3.65 (5H, m, H-1′b, H-1′′, H-6b, H-3), 3.64–3.59 (2H, m, H-6′), 3.52 (1H, dd, J1,2 = 4.0

Hz, J2,3 = 10.0, Hz, H-2), 3.43 (1H, t, J3,4 = 10.0 Hz, H-4) ppm. 13

C-NMR (125 MHz, D2O) δ:

54

103.9 (C-2′′), 103.3 (C-2′), 92.9 (C-1), 80.6 (C-5′), 80.4 (C-5′′), 77.4 (C-4′′), 76.9 (C-3′′), 76.4

(C-3′), 76.3(C-4′), 72.1(C-3), 71.4 (C-4), 71.1(C-5), 71.0 (C-2), 59.9 (C-1′, C-1′′), 34.2 (C-6),

33.5 (C-6′), 33.5 (C-6′′) ppm. HRMS (ESI): calcd. for C18H29Br3O13Na 714.9035, 716.9015

[M + Na]+; found 714.9053, 716.9034.

6,6′,6′′-trichloro-6,6′,6′′-trideoxy-1-kestose (30, 0.032 g, 85%). [α]D = +26.6 (c 1.0,

MeOH). 1H-NMR (500 MHz, D2O) δ: 5.36 (1H, d, J1,2 = 4.0 Hz, H-1), 4.26 (1H, d, J3′,4′ = 8.6

Hz, H-3′), 4.16 (1H, d, J3′′,4′′ = 8.6 Hz, H-3′′), 4.11–4.05 (3H, m, H-4′, H-4′′, H-5), 4.01–3.94

(2H, m, H-5′, H-5′′), 3.89–3.73 (7H, m, H-6, H-6′, H-6′′, H-1′a), 3.71–3.60 (4H, m, H-1′b, H-

1′′, H-3), 3.52 (1H, dd, J1,2 = 4.0 Hz, J2,3 = 9.7 Hz, H-2), 3.47 (1H, t, J3,4 = 9.7 Hz, H-4) ppm.

13C-NMR (125 MHz, D2O) δ: 103.9 (C-2′′), 103.3 (C-2′), 92.8 (C-1), 80.6 (C-5′), 80.5 (C-5′′),

76.6 (C-3′′), 76.4 (C-3′), 76.2 (C-4′′), 75.3 (C-4′), 72.2 (C-3), 71.6 (C-4), 71.0 (C-5), 70.1 (C-

2), 60.2 (C-1′), 60.0 (C-1′′), 45.4 (C-6′′), 45.2 (C-6′), 44.3 (C-6) ppm. HRMS (ESI): calcd. for

C18H29Cl3O13Na 581.0571, 583.0542 [M + Na]+; found 581.0587, 583.0568.

55

2.3.7 1H

and

13C NMR Literature Comparison of Halodeoxysucrose Derivatives with Observation Data

Table 3 1H

and

13C NMR comparison of 6′-bromo-6′-deoxysucrose heptaacetate (2) with literature data

Assign

ment

2

CAS: 951768-18-6

(δ in ppm and J in Hz)

Ref9a

Observed

δ 1H

(400 MHz in CDCl3)

δ 13

C

(100 MHz, CDCl3)

δ 1H

(270 MHz, CDCl3)

δ 13

C

(67.5 MHz, CDCl3)

1 5.75

(d, J = 3.6 Hz) 89.8 5.65 (d) 90.3

2 4.87 (dd) 70.2 4.88 (dd) 70.1

3 5.45

(t, J = 9.9 Hz) 69.5 5.50–5.40 (m) 69.3

4 5.11

(t, J = 9.7 Hz) 70.6 5.06 (t) 68.1

5 4.30 (m) 68.8 4.35–4.24 (m) 68.5

6a 4.36 (dd) 63.4

4.24–4.17 (m) 62.0

6b 4.30 (dd 4.17–4.09 (m)

1′a 4.19 (m) 63.0 4.24–4.17 (m) 62.2

1′b

2′ – 104.0 – 104.3

3′ 5.44 (d, J = 5.9 Hz) 75.7 5.50–5.40 (m) 76.0

4′ 5.42 (t, J = 5.7 Hz 74.8 5.37 (t) 77.1

5′ 4.21 (dd, J = 5.4 Hz, J = 10.6 Hz) 79.1 4.35–4.24 (m) 81.1

6′a 3.60 (dd) 31.2 3.62 (d) 31.3

6′b 3.42 (dd)

J1,2 3.7

–

3.6

– J2,3 10.4 10.2

J3,4 – 9.7

56

Assign

ment

2 (con’t)

CAS: 951768-18-6

(δ in ppm and J in Hz)

Ref9a

Observed

δ 1H

(400 MHz in CDCl3)

δ 13

C

(100 MHz, CDCl3)

δ 1H

(270 MHz, CDCl3)

δ 13

C

(67.5 MHz, CDCl3)

J4,5 –

–

9.7

–

J5,6a 4.4

– J5,6b 5.6

J6a,6b 12.1 or 11.9

J3′,4′ – 5.1

J4′,5′ 5.1

J5′,6′a 2.7 6.6

J5′,6′b 4.4

J,6′a,6′b 11.6 or 11.5 –

CH3 2.17 (s), 2.13 (s), 2.12 (s), 2.11 (s),

2.10 (s), 2.07 (s), 2.02 (s) 20.5, 20.7, 20.7, 20.6, 20.6

2.18 (s). 2.13–2.10 (m), 2.05 (s), 2.02

(s)

20.6, 20.6, 20.5, 20.4, 20.4,

20.4

C=O – 170.5, 170.1, 170.0, 169.9,

169.6, 169.3 –

170.5, 170.0, 169.9, 169.8,

169.7, 169.4

9 Andrade, M. M., et al. Eur. J. Org. Chem. 2007, 7, 3655–3668. a

6′-bromo-6′-deoxysucrose heptaacetate (3) was misinterpreted in this report. In

present study, it was then revised as 6-bromo-6-deoxysucrose heptaacetate (4)

57

Table 4 1H

and

13C NMR comparison of 6-bromo-6-deoxysucrose heptaacetate (3) with literature data

Assign

ment

3

CAS: 54429-71-9 (δ in ppm and J in Hz)

Ref14

Observed

δ 1H (CDCl3) δ

13C

a δ

1H (270 MHz, CDCl3) δ

13C (67.5 MHz,CDCl3)

1

–

–

5.74 (d) 89.8

2 4.87 (dd) 70.1

3 5.49–5.40 (m) 69.4

4 5.11 (t) 70.6

5 4.39–4.25 (m) 68.8

6a 3.54 (m)

3.60 (dd) 31.1

6b 3.42 (dd)

1′a

–

4.20 (s) 63.0 1′b

2′ – 103.9

3′ 5.49–5.40 (m) 75.7

4′ 5.49–5.40 (m) 74.8

5′ 4.25–4.21 (m) 79.0

6′a 4.39–4.25 (m) 63.3

6′b

J1,2 3.6

–

J2,3 10.6

J3,4 9.6

J4,5 9.6

J5,6a 3.0

J5,6b 4.6

J6a,6b 11.5

CH3 2.1 (m) 2.17 (s), 2.12–2.10 (m), 2.07 (s), 2.02 (s) 20.6, 20.6, 20.6, 20.5, 20.5

C=O – – 170.4, 170.0, 169.9, 169.8, 169.6, 169.2 14

Castro, B.; Chapleur, Y.; Gross, B. Carbohydr. Res. 1974, 36, 412–419. a

No previous assignment

58

Table 5 1H

and

13C NMR comparison of 6,6′-dibromo-6,6′-dideoxysucrose hexaacetate (4) with literature data

Assign

ment

4

CAS: 54484-76-3

(δ in ppm and J in Hz)

Ref14

Ref13

Ref10

Ref59

Ref60

Observed

δ 1H

(CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(400

MHz,

DMSO-

d6)

δ 13

C

(100 MHz,

DMSO-d6)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(90 MHZ,

C6D6)

δ 13

Ca

–

δ 1H

(270 MHz,

CDCl3)

δ 13

C

(67.5 MHz,

CDCl3)

1

– –

5.57 (d)

–

5.63 (d,

J = 3.2

Hz)

90.0 5.65 (d, J =

3.6 Hz)

170.09,

170.03,

169.48,

169.35,

104.43,

90.36,

81.09,

77.06,

76.14,

70.61,

70.18,

69.28,

69.19,

62.38,

31.61,

31.14,

20.75,

20.63,

20.59,

20.54,

20.52,

5.78 (d)

–

5.68 (d) 90.3

2 4.85 (q) 4.82

(dd) 69.3

4.84 (dd, J

= 10.4, 3.7

Hz)

4.97 (dd) 4.88 (dd) 70.1

3 5.43 (q)

5.31–

5.35

(m)

68.8

5.41 (dd, J

= 10.2, 9.5

Hz)

5.72 (t) 5.50–5.37

(m) 69.2

4 5.04 (t)

4.99 (t,

J = 9.7

Hz)

69.6 or 68.8 5.03 (t, J =

9.7 Hz) 5.22 (t) 5.07 (t) 70.5

5 –

4.16–

4.25

(m)

–

4.29 (ddd, J

= 10.0, 5.3,

2.7 Hz) –

4.37–4.28

(m) 69.1

59

Assign

ment

4 (con’t)

CAS: 54484-76-3

(δ in ppm and J in Hz)

Ref14

Ref13

Ref10

Ref59

Ref60

Observed

δ 1H

(CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(400

MHz,

DMSO-

d6)

δ 13

C

(100 MHz,

DMSO-d6)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(90 MHZ,

C6D6)

δ 13

Ca

–

δ 1H

(270 MHz,

CDCl3)

δ 13

C

(67.5 MHz,

CDCl3)

6a 3.54 (m)

– – –

3.58–

3.78

(m)

32.6

3.49 (dd , J

= 11.5, 2.7

Hz)

20.48

–

–

3.52 (dd)

31.1

6b

–

3.37(dd, J =

11.6, 5.4

Hz)

3.40 (dd)

1′a 4.16–

4.25

(m)

62.3

4.22 (d, J =

12.5 Hz) 4.44 (d)

4.24 (s) 62.3

1′b 4.19 (d, J =

12.4 Hz) 4.23 (d)

2′ – 103.4 – – – 104.3

3′

5.40 (d,

J = 6.4

Hz)

75.8 5.39 (d, J =

5.1 Hz) 5.60 (d)

5.50–5.37

(m) 76.1

4′

5.31–

5.35

(m)

76.0 5.37 (t, J =

5.0 Hz)

5.46 (t)

5.50–5.37

(m) 77.0

5′

4.29–

4.33

(m)

79.5 4.25 (td, J =

6.7, 4.8 Hz) –

4.37–4.28

(m) 81.0

60

Assign

ment

4 (con’t)

CAS: 54484-76-3

(δ in ppm and J in Hz)

(con’t)

Ref14

Ref13

Ref10

Ref59

Ref60

Observed

δ 1H

(CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(400

MHz,

DMSO-

d6)

δ 13

C

(100 MHz,

DMSO-d6)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(90 MHZ,

C6D6)

δ 13

Ca

–

δ 1H

(270 MHz,

CDCl3)

δ 13

C

(67.5 MHz,

CDCl3)

6′a 3.54 (m)

–

–

–

3.58–

3.78 (m) 33.0

3.62 (d, J =

6.6 Hz)

–

–

3.65 (d) 31.5 6′b

J1,2

–

3.5 3.3

– –

4.0 3.6

–

J2,3 10.0 10.5 10.0 10.6

J3,4 10.0

–

10.0 9.7

J4,5 9.5 10.0 9.7

J5,6a

–

–

3.0

J5,6b 5.6

J6a,6b 11.5

J1′a,1′b 12.5

– J3′,4′ 5.0

J4′,5′ 5.0

J5′,6′ – 6.6

61

Assign

ment

4 (con’t)

CAS: 54484-76-3

(δ in ppm and J in Hz)

(con’t)

Ref14

Ref13

Ref10

Ref59

Ref60

Observed

δ 1H

(CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(400

MHz,

DMSO-

d6)

δ 13

C

(100 MHz,

DMSO-d6)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(90 MHZ,

C6D6)

δ 13

Ca

–

δ 1H

(270 MHz,

CDCl3)

δ 13

C

(67.5 MHz,

CDCl3)

CH3 2.1 (m)

–

7.84-7.99

1.95–

2.06 (m)

20.5, 20.4,

20.3, 20.2,

20.1

2.14(s),

2.08 (s),

2.08 (s),

2.07 (s),

2.03 (s),

1.99 (s) – – –

2.18 (s),

2.16–2.09

(m), 2.07

(s), 2.02 (s)

20.6, 20.5,

20.5, 20.4,

20.4, 20.4

C=O – – – 169.6,

169.2, 169.1 – –

170.0,

169.9,

169.9,

169.7,

169.4 10

Barros, M. T.; Petrova, K. T.; Correia-da-Silva, P.; Potewar, T. M.; Correia-Da-Silva, P.; Potewar, T. M.; Correia-da-Silvaa, P.; Potewar, T. M.

Green Chem. 2011, 13, 1897–1906. 13

Khan, R.; Lal Bhardwaj, C.; Mufti, K. S.; Jenner, M. R. Carbohydr. Res. 1980, 78, 185–189. 14

Castro, B.;

Chapleur, Y.; Gross, B. Carbohydr. Res. 1974, 36, 412–419. 59

Lees, W. J.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1860–1869. 60

Hough, L.;

Sinchareonkul, L. V.; Richardson, A. C.; Akhtar, F.; Drew, M. G. B. Carbohydr. Res. 1988, 174, 145–160. a

Not assigned.

62

Table 6 1H

and

13C NMR comparison of 6′-chloro-6′-deoxysucrose heptaacetate (6) with

literature data

Assignment

6

CAS: 50271-00-6

(δ in ppm and J in Hz)

Ref5 Ref

11 Observed

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(270 MHz,CDCl3)

δ 13

Ca

(67.5

MHz,CDCl3)

1

–

–

5.64 (d)

–

5.64 (d) 90.3

2 4.86 (dd) 4.87 (dd) 70.2

3 – 5.49–5.40 (m) 69.4

4 5.05 (t) 5.06 (t) 68.2

5

–

4.33–4.25 (m) 68.5

6a 4.25–4.17 (m) 62.0

6b 4.13 (dd)

1′a 4.25–4.17 (m) 62.4

1′b

2′ – 104.4

3′ 5.45 (q) 5.5–5.3

(m) 5.49–5.40 (m) 76.0

4′ 4.35 (t) 5.5–5.3

(m) 5.37 (t) 76.5

5′

–

4.25–4.17 (m) 81.3

6′a 3.76 (d) 3.77 (d) 43.9

6′b

J1,2 3.5 3.6

–

J2,3 10.0 10.2

J3,4 9.5 9.9

J4,5 9.5 9.9

J5,6b

–

2.0

J6a,6b 12.2

J3′,4′ 5.0 5.1

J4′,5′ 5.0 5.1

J5′,6′

–

6.6

CH3

2.18 (s), 2.15–2.08

(m), 2.05 (s), 2.02

(s)

20.7, 20.6, 20.6,

20.5, 20.4

C=O – –

170.6, 170.0,

169.9, 169.8,

169.5 5 Khan, R.; Jenner, M. R.; Mufti, K. S. Carbohydr. Res. 1975, 39, 253–262.

11 Ballard, J. M.;

Hough, L.; Richardson, A. C.; Fairclough, P. H. J. Chem. Soc., Chem. Commun. 1972, 1524–

1528. a

No previous literature data

63

Table 7 1H

and

13C NMR comparison of 6-chloro-6-deoxysucrose heptaacetate (7) with

literature data

Assignment

7

CAS: 54429-73-1

(δ in ppm and J in Hz)

Ref5 Ref

14 Observed

δ 1H

(100

MHz,

CDCl3)

δ 13

Ca

–

δ 1H

(CDCl3)

δ 13

Ca

–

δ 1H

(270

MHz,CDCl3)

δ 13

Ca

(67.5 MHz,CDCl3)

1 5.74

(d)

–

–

–

5.74 (d) 89.6

2 4.85

(q) 4.87 (dd) 70.0

3 5.45

(q) 5.49–5.39 (m) 69.4

4 5.13 (t) 5.14 (t) 69.2

5

–

4.41–4.26 (m) 69.2

6a 3.58 (m)

3.73 (dd) 43.0

6b 3.58 (dd)

1′a

–

4.20 (s) 62.8 1′b

2′ – 103.7

3′ 5.43

(d) 5.49–5.39 (m) 75.5

4′ 5.21 (t) 5.49–5.39 (m) 74.6

5′

–

4.25–4.21 (m) 78.8

6′a 4.41–4.26 (m) 63.1

6′b

J1,2 3.5 3.6

–

J2,3 10.0 10.6

J3,4 9.5 9.7

J4,5 9.5 9.7

J5,6a

–

3.0

J5,6b 4.6

J6a,6b 12.2

J3′,4′ 6.0

J4′,5′ 6.0

CH3 2.2–2.0 2.1 (m)

2.17 (s), 2.13–

2.08 (m), 2.06

(s), 2.02 (s)

20.4, 20.4, 20.4

C=O – – – 170.2, 169.8, 169.7,

169.6, 169.4, 169.1 5 Khan, R.; Jenner, M. R.; Mufti, K. S. Carbohydr. Res. 1975, 39, 253–262.

14 Castro, B.;

Chapleur, Y.; Gross, B. Carbohydr. Res. 1974, 36, 412–419. a

No previous literature data

64

Table 8 1H

and

13C NMR comparison of 6,6′-dichloro-6,6′-dideoxysucrose hexaacetate (8)

with literature data

Assignment

8

CAS: 40984-14-3

(δ in ppm and J in Hz)

Ref4 Observed

δ 1H

(100

MHz,

acetone-

d6)

δ 13

Ca

–

δ 1H

(270 MHz,CDCl3)

δ 13

C

(67.5 MHz,CDCl3)

1 5.7 (d)

–

5.68 (d) 90.3

2 4.86 (q) 4.87 (dd) 70.1

3 5.46 (t) 5.50–5.35 (m) 69.4

4 5.1 (t) 5.10 (t) 69.5

5

–

4.40–4.31 (m) 69.6

6a 3.67 (dd) 43.2

6b 3.56 (dd)

1′a 4.23 (s) 62.4

1′b

2′ – 104.4

3′ 5.5 (d) 5.50–5.35 (m) 75.9

4′ 5.42 (t) 5.50–5.35 (m) 76.4

5′ –

4.29–4.24 (m) 81.3

6′ 3.79 (d) 44.0

J1,2 3.5 3.6

–

J2,3 10.0 10.2

J3,4 10.0 9.7

J4,5 10.0 9.7

J5,6a

–

3.0

J5,6b 4.9

J6a,6b 12.2

J3′,4′ 5.5 –

J4′,5′ 5.5

J5′,6′ 6.3

CH3

–

2.18 (s), 2.16–2.08 (m),

2.07 (s), 2.02 (s) 20.7, 20.6, 20.5, 20.5, 20.4

C=O – 170.0, 170.0, 169.7, 169.5,

169.3 4 Hough, L.; Mufti, K. S. Carbohydr. Res. 1972, 25, 497–503.

a No previous literature data

65

Table 9 1H

and

13C NMR comparison of 1′-bromo-1′-deoxysucrose heptaacetate (10) with

literature data

Assignment

10

CAS: –

(δ in ppm and J in Hz)

Refa Observed

δ 1H

–

δ 13

C

–

δ 1H

(500 MHz,CDCl3)

δ 13

C

(125 MHz,CDCl3)

1

– –

5.74–5.71 (m) 89.7

2 4.91 (dd) 69.9

3 5.47–5.40 (m) 69.6

4 5.08 (t) 68.0

5 4.34–4.26 (m) 68.4

6a 4.34–4.26 (m) 61.6

6b 4.16 (d)

1′a 3.61 (d) 33.5

1′b 3.49 (d)

2′ – 103.4

3′ 5.74–5.71 (m) 76.1

4′ 5.47–5.40 (m) 74.0

5′ 4.24–4.20 (m) 78.9

6′a 4.34–4.26 (m) 63.1

6′b

J1,2 4.6

–

J2,3 10.3

J4,5 9.5

J6a,6b 11.5

J1′a,1′b 12.0

CH3

2.17 (s), 2.12 (s), 2.11–

2.09 (m), 2.08 (s), 2.04 (s),

2.02 (s)

20.7, 20.6, 20.6, 20.5, 20.5

C=O – 170.6, 170.4, 170.0, 169.9,

169.7, 169.6, 169.4 aNo previous literature data

66

Table 10 1H

and

13C NMR comparison of 1′-chloro-1′-deoxysucrose heptaacetate (11) with

literature data

Assignment

11

CAS: 64644-61-7

(δ in ppm and J in Hz)

Ref8 Ref

68 Observed

δ 1H

(60 MHz in

CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz in

CDCl3)

δ 13

Ca

–

δ 1H

(500

MHz,CDCl3)

δ 13

C

(125

MHz,CDCl3)

1 –

–

5.71 (d)

–

5.72 (d) 89.5

2 4.89 (dd) 4.89 (q) 4.91 (dd) 69.9

3 5.45 (t) 5.44 (t) 5.46–5.41 (m) 69.6

4 5.05 (t) 5.06 (t) 5.08 (t) 68.0

5 4.15-4.40 (m)

–

4.36–4.27 (m) 68.4

6a 4.15-4.40 (m)

4.36–4.27 (m) 61.5

6b 4.16 (dd)

1′a 3.48 (d) 3.74 (d) 45.3

1′b 3.75 (d) 3.57 (d)

2′ – – – 103.9

3′ 5.65 (d)

5.68 (d) 5.69 (d) 75.4

4′ 5.40(t) 5.4 (t) 5.46–5.41 (m) 73.9

5′ 4.15-4.40 (m)

–

4.22 (td) 78.9

6′a 4.15-4.40 (m) 4.36–4.27 (m) 63.0

6′b

J1,2 4.1 3.5 4.0

–

J2,3 10 10.0 10.3

J3,4 9 or 10 9.5

J4,5 9 9.5 9.7

J5,6a

– –

J5,6b 2.9

J6a,6b 13.2

J1′a,1′b 12 12.0

J3′,4′ 6.5 or 7 6.5 6.3

J4′,5′ 7 6.5 6.4

J5′,6′ – 3.6

CH3

1.98 (s), 1.99

(s), 2.00 (s),

2.01(s), 2.08

(s)

7.84-8.01

2.18 (s), 2.13 (s),

2.10 (s), 2.07 (s),

2.04 (s), 2.02 (s)

20.6, 20.6,

20.6, 20.5

C=O – – –

170.6, 170.4,

170.0, 169.9,

169.8, 169.6,

169.4 8 Gus, R. D. G.; Guthrie, R. D.; Jenkins, I. D.; Watters, J. J. Autr. J. Chem. 1980, 33, 2487–

2497. 68

Khan, R.; Jenner, M. R.; Lindseth, H. Carbohydr. Res. 1980, 78, 173–183. a

No

previous literature data

67

Table 11 1H

and

13C NMR comparison of sucralose pentaacetate (13) with literature data

Assignment

13

CAS: 55832-20-7

(δ in ppm and J in Hz)

Ref5 Ref

79 Observed

δ 1H

(100 MHz in

CDCl3)

δ 13

Ca

–

δ 1H

(100

MHz in

C6D6)

δ 13

Ca

–

δ 1H

(500 MHz,CDCl3)

δ 13

C

(125

MHz,CDCl3)

1 5.7 (d)

–

5.79 (d)

–

5.68 (d) 90.6

2 5.5–5.23 (m) 5.56 (q) 5.31–5.28 (m) 67.9

3 5.5–5.23 (m) 5.68 (q) 5.31–5.28 (m) 66.8

4 5.5–5.23 (m)

–

4.60–4.54 (m) 58.9

5

–

4.60–4.54 (m) 67.7

6a 4.28–4.21 (m) 63.5

6b

1′a 3.71 (d) 44.4

1′b 3.60 (d)

2′ – 104.3

3′ 5.68 (d) 5.78 (d) 5.70 (d) 75.8

4′ 5.4 (t) 5.38 (t) 5.41 (t) 76.0

5′

–

4.28–4.21 (m) 80.7

6′a 3.77 (d) 43.8

6′b

J1,2 3.0 4.0 1.7

–

J2,3

–

11.0

–

J3,4 3.5

J4,5

– J5,6a

J5,6b

J1′a,1′b 12.0

J3′,4′ 6.0 7.0 6.3

J4′,5′ 6.0 7.0 6.3

J5′,6′ – – 5.7

CH3 7.84-7.95 7.85–7.92

2.15(s), 2.14(s),

2.12 (s), 2.10 (s),

2.10 (s)

20.8, 20.7,

20.7, 20.5

C=O – – – 170.4, 170.0,

169.8, 169.6 5 Khan, R.; Jenner, M. R.; Mufti, K. S. Carbohydr. Res. 1975, 39, 253–262.

79 Fairclough, P.

H.; Hough, L.; Richardson, A. C. Carbohydr. Res. 1975, 40, 285–298. a

No previous literature

data.

68

Table 12 1H

and

13C NMR comparison of 6′-bromo-6′-deoxysucrose (14) with literature data

Assignment

14

CAS: –

(δ in ppm and J in Hz)

Refa Observed

δ 1H

–

δ 13

C

–

δ 1H

(500 MHz, D2O )

δ 13

C

(125 MHz, D2O )

1

– –

5.34 (d) 92.4

2 3.50 (dd) 71.1

3 3.73–3.66 (m) 72.6

4 3.36 (t) 69.5

5 3.84–3.78 (m) 72.7

6a 3.84–3.78 (m) 60.5

6b 3.73–3.66 (m)

1′ 3.64 (brs) 61.0

2′ – 103.9

3′ 4.17 (d) 76.6

4′ 4.04 (t) 77.0

5′ 4.02–3.98 (m) 80.8

6′ 3.73–3.66 (m) 33.2

J1,2 4.0

–

J2,3 9.7

J3,4 9.7

J4,5 9.7

J3′,4′ 8.0

J4′,5′ 8.0 a No previous literature data

69

Table 13 1H

and

13C NMR comparison of 6-bromo-6-deoxysucrose (15) with literature data

Assignment

15

CAS: 99789-72-7

(δ in ppm and J in Hz)

Ref14

Observed

δ 1H

a

(CDCl3)

δ 13

Cb

–

δ 1H

(500 MHz, D2O )

δ 13

C

(125 MHz, D2O)

1

–

–

5.38 (d) 92.1

2 3.55 (dd) 71.0

3 3.76–3.71 (m) 72.1

4 3.47 (t) 71.2

5 3.99 (dt) 70.7

6a 3.54 (m) 3.76–3.71 (m) 33.4

6b

1′

–

3.63 (brs) 61.4

2′ – 103.7

3′ 4.18 (d) 76.2

4′ 4.05 (t) 74.0

5′ 3.86 (td) 81.3

6′ 3.81–3.76 (m) 62.5

J1,2 4.0

–

J2,3 9.7

J3,4 9.7

J4,5 9.7

J5,6 3.4

J3′,4′ 8.6

J4′,5′ 8.6

J5′,6′ 3.4

CH3 2.1 (m) – 14

Castro, B.; Chapleur, Y.; Gross, B. Carbohydr. Res. 1974, 36, 412–419. a

Assigned as 6-

bromo-6-deoxysucrose heptaacetate (3). b No previous literature data

70

Table 14 1H

and

13C NMR comparison of 6,6′-dibromo-6,6′-dideoxysucrose (16) with

literature data

Assignment

16

CAS: 33585-15-8

(δ in ppm and J in Hz)

Ref10

Observed

δ 1H

(400 MHz in

D2O)

δ 13

C

(100 MHz, D2O )

δ 1H

(500 MHz, D2O )

δ 13

C

(125 MHz, D2O )

1 5.39 (d, J = 3.9

Hz) 92.4 5.35 (d) 92.3

2 3.56 (dd) 71.0 3.52 (dd) 71.0

3 3.64–3.69 (m) 72.1 3.75–3.69 (m) 72.0

4 3.42 (t, J = 9.4

Hz) 71.5 3.42 (t) 71.4

5 4.01–4.06 (m) 71.2 4.01–4.00 (m) 71.0

6a 3.70–3.79 (m) 33.4

3.69–3.65 (m) 33.3

6b 3.63–3.60 (m)

1′a 3.64–3.69 (m) 61.2

3.64 (brs) 61.1

1′b

2′ – 103.9 – 103.8

3′ 4.20 (d, J = 8.5

Hz)

76.6

4.17 (d) 76.4

4′ 4.11 (t, J = 8.0

Hz) 76.7 4.08 (t) 76.6

5′ 4.01–4.06 (m) 80.5 4.01–4.00 (m) 80.5

6′ 3.70–3.79 (m) 33.4 3.75–3.69 (m) 33.2

J1,2 3.9

–

4.0

–

J2,3 9.9 9.7

J3,4

–

9.5

J4,5 9.5

J3′,4′ 8.6

J4′,5′ 8.6 10

Barros, M. T.; Petrova, K. T.; Correia-da-Silvaa, P.; Potewar, T. M.; Correia-Da-Silva, P.;

Potewar, T. M.; Correia-da-Silvaa, P.; Potewar, T. M. Green Chem. 2011, 13, 1897–1906.

71

Table 15 1H

and

13C NMR comparison of 6′-chloro-6′-deoxysucrose (17) with literature data

Assignment

17

CAS: 50270-99-0

(δ in ppm and J in Hz)

Ref81a

Observed

δ 1H

(D2O)

δ 13

C

(D2O)

δ 1H

(500 MHz, D2O)

δ 13

C

(125 MHz,

D2O)

1 –

104.6, 81.5, 77.1,

76.7, 73.3, 71.7,

70.2, 61.8,

61.1, 45.9

5.33 (d) 92.3

2 3.56 (d) 3.49 (dd) 71.0

3 3.76 (t) 3.73–3.66 (m) 72.5

4 3.43 (t) 3.36 (t)

69.4

5 3.85–3.77 (m) 72.6

6a 3.92–3.82 (m)

3.85–3.77 (m) 60.3

6b 3.73–3.66 (m)

1′a 3.71 (s) 3.64 (brs) 60.9

1′b

2′ – – 103.8

3′ 4.24 (d) 4.17 (d) 76.3

4′ 4.13 (t) 4.06 (t) 75.9

5′ 4.04 (dt) 3.97 (dd) 80.7

6′a 3.92–3.82 (m) 3.85–3.77 (m) 45.1

6′b

J1,2

–

3.4

–

J2,3 9.6 9.7

J3,4 9.6 9.7

J4,5 9.6 9.7

J3′,4′ 8.3 8.6

J4′,5′ 8.3 8.6

J5′,6′a 5.6 5.7 81

Kakinuma, H.; Yuasa, H.; Hashimoto, H. Carbohydr. Res. 1998, 312, 103–115. a[1-

2H]-6′-

chloro-6′deoxysucrose.

72

Table 16 1H

and

13C NMR comparison of 6-chloro-6-deoxysucrose (18) with literature data

Assignment

18

CAS: 40984-18-7

(δ in ppm and J in Hz)

Ref4 Ref

5 Observed

δ 1H

a

–

δ 13

Cb

–

δ 1H

c

(100 MHz,

CDCl3)

δ 13

Cb

–

δ 1H

(500 MHz,

D2O)

δ 13

C

(125 MHz,

D2O)

1

– –

5.74 (d)

–

5.36 (d) 92.1

2 4.85 (q) 3.52 (dd) 71.0

3 5.45 (q) 3.72–3.69 (m) 72.1

4 5.13 (t) 3.47 (t) 69.9

5

–

4.05 (dt) 71.3

6a 3.86–3.82 (m) 44.2

6b

1′ 3.61 (brs) 61.3

2′ – 103.7

3′ 5.43 (d) 4.16 (d) 76.1

4′ 5.21 (t) 4.00 (t) 74.0

5′ –

3.86–3.82 (m) 81.3

6′ 3.77 (d) 62.5

J1,2 3.5 3.4

–

J2,3 10.0 9.7

J3,4 9.5 9.7

J4,5 9.5 9.7

J5,6a – 3.2

J5,6b

J3′,4′ 6.0 8.6

J4′,5′ 6.0 8.6

J5′,6′ – 2.9

CH3 7.8-8.0 – 4

Hough, L.; Mufti, K. S. Carbohydr. Res. 1972, 25, 497–503. 5 Khan, R.; Jenner, M. R.;

Mufti, K. S. Carbohydr. Res. 1975, 39, 253–262. a

Not assigned. b No previous literature data.

c Assigned as 6-chloro-6-deoxysucrose heptaacetate (6).

73

Table 17 1H

and

13C NMR comparison of 6,6′-dichloro-6,6′-dideoxysucrose (19) with

literature data

Assignment

19

CAS: 40984-16-5

(δ in ppm and J in Hz)

Ref10

Ref69

Observed

δ 1H

(400

MHz,D2O)

δ 13

C

(100 MHz,

D2O)

δ 1H

a

–

δ 13

C

(D2O)

δ 1H

(500

MHz,D2O)

δ 13

C

(125 MHz,

D2O)

1 5.31 (d, J =

3.6 Hz) 92.7

–

93.0 5.39 (d) 92.3

2 3.47 (dd) 71.4 73.2 3.55 (dd) 71.0

3 3.67 (t) 72.0 72.6 3.74 (t) 72.1

4 3.40 (t) 70.5 71.4 3.48 (t) 70.1

5 4.04 (m) 72.5 72.0 4.14–4.08

(m) 71.6

6a 3.78 (m) 44.7 45.4

3.91–3.84

(m) 44.3

6b

1′a 3.59 (d, J =

1.7 Hz) 61.5 62.4 3.67 (brs) 61.2

1′b

2′ – 104.3 104.8 – 103.9

3′ 4.13 (d) 76.7 77.7 4.21 (d) 76.3

4′ 3.92 (m) 76.0 81.6 4.14–4.08

(m) 75.6

5′ 4.04 (m) 81.0 76.9 4.00 (td) 80.7

6′ 3.78 (m) 45.5 46.2 3.84–3.78

(m) 45.2

J1,2 3.7

– –

4.0

–

J2,3 9.9 9.7

J3,4 9.5 or 9.6 9.7

J4,5 9.5 9.7

J3′,4′ 8.6 8.6

J4′,5′ –

8.0

J5′,6′ 3.4 10

Barros, M. T.; Petrova, K. T.; Correia-da-Silva, P.; Potewar, T. M.; Correia-Da-Silva, P.;

Potewar, T. M.; Correia-da-Silvaa, P.; Potewar, T. M. Green Chem. 2011, 13, 1897–1906. 69

Hough, L.; Phadnis, S. P.; Tarelli, E. Carbohydr. Res. 1976, 47, 151–154. a

Not assigned

74

Table 18 1H

and

13C NMR comparison of 1′-bromo-1′-deoxysucrose (20) with literature data

Assignment

20

CAS: –

(δ in ppm and J in Hz)

ref.a Observed

δ 1H

–

δ 13

C

–

δ 1H

(500 MHz, D2O)

δ 13

C

(125 MHz, D2O)

1

– –

5.40 (d) 92.7

2 3.52 (dd) 71.1

3 3.73–3.69 (m) 72.7

4 3.44 (t) 69.4

5 3.82–3.76 (m) 72.7

6 3.82–3.76 (m) 60.2

1′a 3.73–3.69 (m) 32.6

1′b 3.63 (brs)

2′ – 102.8

3′ 4.42 (d) 77.0

4′ 4.01 (t) 73.6

5′ 3.87 (td) 81.9

6′ 3.82–3.76 (m) 62.1

J1,2 4.0

–

J2,3 9.7

J3,4 9.7

J4,5 9.7

J3′,4′ 8.6

J4′,5′ 8.6

J5′,6′ 2.9 a No previous literature data

75

Table 19 1H

and

13C NMR comparison of 1′-chloro-1′-deoxysucrose (21) with literature data

Assign

ment

21

CAS: 64644-62-8

(δ in ppm and J in Hz)

Ref8 Ref

82 Observed

δ 1H (60 MHz,D2O) δ

13C (D2O)

δ 1H (500 MHz in DMSO-

d5)

δ 13

Cd

– δ

1H (500 MHz, D2O) δ

13C (125 MHz,D2O)

1 5.44 (d) 93.3

5.18

–

5.40 (d) 92.5

2

3.45-4.20 (m, 20H)

71.8 3.16 3.52 (dd) 70.9

3 73.4 3.41 3.71 (t) 72.5

4 70.2 3.11 3.43 (t) 69.2

5 72.5 3.63 3.81 (dd) 72.5

6a 61.0

3.52–3.60a

3.80–3.77 (m)

60.0 6b 3.52–3.60

a

1′a 44.6

3.67 3.77 (brs) 43.8

1′b 3.63

2′ 103.9

– – 103.1

3′ 77.2 4.01 4.36 (d) 76.2

4′ 74.4 3.76 4.01 (t) 73.4

5′ 82.6

3.52–3.60a 3.88 (td) 81.7

6′a 62.8

3.52–3.60a

3.80–3.77 (m) 61.9 6′b 3.48

O2

– –

4.77

– –

O3 4.76

O4 4.77

O6 4.38

O3 4.94

O4 5.30

O6 4.45

76

Assign

ment

21 CAS: 64644-62-8 (δ in ppm and J in Hz) (con’t)

Ref8 Ref

82 Observed

δ 1H (60 MHz,D2O) δ

13C (D2O)

δ 1H (500 MHz in

DMSO-d5)

δ 13

Cd

– δ

1H (500 MHz, D2O) δ

13C (125 MHz,D2O)

J1,2 –

–

3.7

–

4.0

–

J2,3 3 9.8 9.7

J3,4

–

8.9 9.7

J4,5 10.0 9.7

J5,6a 2.2 2.9

J5,6b 4.6 5.7

J6a,6b b

J1′a,1′b 12.2c

J3′,4′ 8.6 8.6

J4′,5′ 7.8 8.6

J5′,6′a b 3.4

J5′,6′b 4.8

J6′a,6′b 10.9c

–

J2,OH2 6.2

J3,OH3 5.0

J4,OH4 5.6

J6a,OH6 5.2

J6b,OH6 6.0

J3′,OH3′ 8.0

J4′,OH4′ 5.8

J6′a,OH6′ b

J6′b,OH6′ b

8 Gus, R. D. G.; Guthrie, R. D.; Jenkins, I. D.; Watters, J. J. Autr. J. Chem. 1980, 33, 28487–2497.

82 Christofides, J. C.; Davies, D. B.; Martin, J. A.;

Rathbone, E. B. J. Am. Chem. Soc. 1986, 108, 5738–5743. a

Due to overlap of signals, chemical shifts could not be determined accurately. b

Due to

overlap of signals, couplings could not be determined accurately. c Magnitudes but not signs were determined.

d Not assigned

77

2.3.8 1H

and

13C NMR Literature Comparison of Halodeoxy-1-kestose Derivatives with Observation Data

Table 20 1H and

13C NMR literature comparison with observation data of 1-kestose undecaacetate (23) and per-O-acetylated trihalogenated 1-kestose

derivatives (24 and 25)

Assign

ment

23

CAS: 25101-98-8

(δ in ppm and J in Hz)

24

CAS: -

(δ in ppm and J in Hz)

25

CAS: -

(δ in ppm and J in Hz)

Ref.76

Ref.80

Observed Observed Observed

δ 1H

(CDCl3)

δ 13

C

(100.6

MHz,

CDCl3)

δ 1H

(100 or 220

MHz,

CDCl3)

δ 13

Ca

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

1 5.71 (d) 90.3 (d) 5.71

—

5.75 (d) 89.2

5.67 (d) 90.1 5.70–5.66 (m) 90.0

2 4.88 (dd) 70.3 (d) 4.88 4.91 (dd) 70.0 4.95 (dd) 69.7 4.93 (dd) 69.7

3 5.42 (t) 70.1 (d) 5.42 5.42 (t) 69.8 5.48–5.42 (m) 69.3 5.48–5.41 (m) 69.4

4 5.22 (t) 68.0 (d) 5.04 5.08 (t) 68.2 5.03 (t) 70.8 5.06 (t) 69.7

5

4.40–4.20

(m,

overlap

13H

signals)

69.0 (d)

4.42–4.07

(11H

signals)

4.39–4.33

(m) 68.2 4.35–4.28 (m) 69.3 4.38-4.32 (m) 69.8

6a

59.7 (t)

4.42–4.07

(11H

signals)

4.33–4.24

(m)

61.7

3.54 (dd)

31.1

3.68 (dd)

43.2

6b

4.42–4.07

(11H

signals)

4.20–4.14

(m) 3.40 (dd) 3.57dd)

78

Assign

ment

23 (con’t)

CAS: 25101-98-8

(δ in ppm and J in Hz)

24 (con’t)

CAS: -

(δ in ppm and J in Hz)

25 (con’t)

CAS: -

(δ in ppm and J in Hz)

Ref.76

Ref.80

Observed Observed Observed

δ 1H

(CDCl3)

δ 13

C

(100.6

MHz,

CDCl3)

δ 1H

(100 or

220

MHz,

CDCl3)

δ 13

Ca

δ 1H

(500

MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

1′a 63.7 (t)

3.71

—

3.69 (d) 62.2

3.79 (d) 61.5 3.75 (s) 61.6

1′b 3.62 3.63 (d) 3.73 (d)

2′ — 104.3 (s) — — 103.4 — 103.8 — 103.8

3′ 5.37 (d) 76.2 (d) 5.46 5.69 (d) 74.9 5.70 (d) 75.8 5.70–5.66

( m) 75.5

4′ a 75.3 (d) 5.32 5.46 (t) 73.7 5.48–5.42 (m) 76.4 5.48–5.41 (m) 75.7

5′

4.40–4.20

(m,

overlap

13H

signals)

78.1 (d)

4.42–

4.07

(11H

signals)

4.24–

4.20 (m) 77.8 4.35–4.28 (m) 79.8 4.28–4.22 (m) 79.9

6′ 62.5 (t)

4.42–

4.07

(11H

signals)

4.33–

4.24 (m) 63.2 3.66 (d) 32.0 3.82–3.78 (m) 44.4

1′′ 64.4 (t)

4.42–

4.07

(11H

signals)

4.24–

4.20 (m) 62.7 4.24 (s) 62.0 4.28–4.22 (m) 62.2

79

Assign

ment

23 (con’t)

CAS: 25101-98-8

(δ in ppm and J in Hz)

24 (con’t)

CAS: -

(δ in ppm and J in Hz)

25 (con’t)

CAS: -

(δ in ppm and J in Hz)

Ref.76

Ref.80

Observed Observed Observed

δ 1H

(CDCl3)

δ 13

C

(100.6

MHz,

CDCl3)

δ 1H

(100 or

220

MHz,

CDCl3)

δ 13

Ca

δ 1H

(500

MHz,

CDCl3)

δ 13

C

(125

MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

2′′ — 103.1 (s) —

—

— 102.9 — 103.1 — 103.1

3′′ 5.46 (d) 76.6 (d) 5.68 5.48 (d) 76.5 5.50 (d) 77.2 5.50 (d) 76.9

4′′ a 76.0 (d) 5.22 5.34 (t) 75.5 5.32 (t) 78.1 5.33 (t) 77.2

5′′ 4.40–4.20

(m,

overlap

13H

signals)

79.1 (d)

4.42–

4.07

(11H

signals)

4.20–

4.14 (m) 78.4 4.22–4.19 (m) 80.7 4.17 (q) 80.6

6′′ 62.9 (t)

4.42–

4.07

(11H

signals)

4.39–

4.33 (m) 63.7 3.69 (d) 32.7 3.82–3.78 (m) 44.6

J1,2 3.4

—

3.9 3.4

—

4.0

—

3.7

—

J2,3 10.2 9.0 10.3 10.3 10.6

J3,4 10.2 9.4 9.7 9.7 9.7

J4,5

—

10.0

—

2.3 2.3

J5,6a

—

— —

J5,6b 6.3 5.7

J6a,6b 11.5 12.0

J1′a,1′b 9.2 10.3 —

J3′,4′ 8.6 7.0 8.0 8.0 —

80

Assign

ment

23 (con’t)

CAS: 25101-98-8 (δ in ppm and J in Hz)

24 (con’t)

CAS: - (δ in ppm and J in Hz)

25 (con’t)

CAS: - (δ in ppm and J in Hz)

Ref.76

Ref.80

Observed Observed Observed

δ 1H

(CDCl3)

δ 13

C

(100.6 MHz,

CDCl3)

δ 1H

(100 or 220

MHz, CDCl3)

δ 13

Ca

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

δ 1H

(500 MHz,

CDCl3)

δ 13

C

(125 MHz,

CDCl3)

J4′,5′ —

—

6.0

—

—

—

—

—

—

—

J5′,6′ 6.9

J3′′,4′′ 8.6 8.0 6.9 6.3 6.3

J4′′,5′′ —

8.0 —

— —

J5′′,6′′ 7.4 6.5

CH3

2.09 (s),

2.12 (s),

2.11 (s),

2.10 (s),

2.09 (s),

2.08 (s),

2.07 (s),

2.05 (s),

2.02 (s),

2.00 (s)

21.5–20.8

2.12

2.09

2.08

2.07

1.99

1.97

2.19–2.14

(m),

2.13–2.12

(m),

2.11–2.09

(m),

2.06 (s),

2.04 (s),

2.01 (s)

20.8,

20.7,

20.7,

20.6,

20.6,

20.5

2.16–2.15

(m),

2.14 (s),

2.11 (s),

2.09-2.07 (m),

2.02 (s)

20.8, 20.8,

20.7, 20.8,

20.6, 20.5,

20.5, 20.5

2.16 (s),

2.15–2.13 (m),

2.11 (s),

2.09 (s),

2.08 (s),

2.07 (s),

2.02 (s)

20.8,

20.7,

20.7,

20.6,

20.5,

20.5

C=O — 170.4–169.7 — —

170.7,

170.6,

170.5,

170.1,

169.9,

169.7,

169.6

—

170.2,

170.0,

170.0,

169.8,

169.5

—

170.2,

170.1,

170.0,

170.0,

169.7,

169.5

76 Pejin, B.; Iodice, C.; Tommonaro, G.; Sabovljevic, M.; Bianco, A.; Tesevic, V.; Vajs, V.; De Rosa, S. Nat. Prod. Res. 2012, 26, 209–215.

80 Binkley, W. W.; Horton, D.; Bhacca, N. S. Carbohydr. Res. 1969, 155, 245–258.

a Not assign.

81

Table 21 1H and

13C NMR literature comparison with observation data of 1-kestose (22) and trihalogenated 1-kestose derivatives (29 and 30)

Assign-

ment

22

CAS 562-68-5

(δ in ppm and J in Hz)

29

CAS: -

(δ in ppm and J in Hz)

30

CAS: -

(δ in ppm and J in Hz)

Ref.71

Observed

Observed

Observed

1H in ppm

(200.13 MHz,

D2O)

13C in ppm

(50.32 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1 5.26 93.73 5.37 (d) 92.9 5.36 (d) 92.9 5.36 (d) 92.8

2 3.38 72.39 3.48 (dd) 71.6 3.52 (dd) 71.0 3.52 (dd) 70.1

3 3.59 73.85 3.70–3.60 (m) 73.0 3.72–3.65 (m) 72.1 3.71–3.60 (m) 72.2

4 3.30 70.48 3.41 (t) 69.6 3.43 (t) 71.4 3.47 (t) 71.6

5 3.64

3.68 73.67 3.79–3.71 (m) 72.8 4.04–3.98 (m) 71.1 4.11–4.05 (m) 71.0

6a 3.63 61.40 3.79–3.71 (m) 60.5

3.79–3.72 (m) 34.2 3.89–3.73 (m) 44.3

6b 3.72–3.65 (m)

1′a 3.54 62.17

3.79–3.71 (m) 61.3

3.87 (d) 59.9

3.89–3.73 (m) 60.2

1′b 3.66 3.70–3.60 (m) 3.72–3.65 (m) 3.71–3.60 (m)

2′ – 104.50 – 103.7 – 103.3 – 103.3

3′ 4.12 77.92 4.22 (d) 77.0 4.27 (d) 76.4 4.26 (d) 76.4

4′ 3.88 75.12 3.98 (t) 74.2 4.09–4.05 (m) 76.3 4.11–4.05 (m) 75.3

5′ 3.67

3.70 82.46 3.83–3.80 (m) 81.6 4.04–3.98 (m) 80.6 4.01–3.94 (m) 80.6

6′a 3.63 63.44 3.79–3.71 (m) 62.6 3.64–3.59 (m) 33.5 3.89–3.73 (m) 45.2

6′b

82

Assign-

ment

22 (con’t)

CAS 562-68-5

(δ in ppm and J in Hz)

29 (con’t)

CAS: -

(δ in ppm and J in Hz)

30 (con’t)

CAS: -

(δ in ppm and J in Hz)

Ref.71

Observed

Observed

Observed

1H in ppm

(200.13 MHz,

D2O)

13C in ppm

(50.32 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1H in ppm

(500 MHz,

D2O)

13C in ppm

(125 MHz,

D2O)

1′′a 3.50 61.70 3.70–3.60 (m) 60.8 3.72–3.65 (m) 59.9 3.71–3.60 (m) 60.0

1′′b 3.59

2′′ – 104.96 – 104.2 – 103.9 – 103.9

3′′ 4.02 77.94 4.13 (d) 77.0 4.16 (d) 76.9 4.16 (d) 76.6

4′′ 3.91 75.74 4.02 (t) 74.9 4.09–4.05 (m) 77.4 4.11–4.05 (m) 76.2

5′′ 3.69 82.36 3.83–3.80 (m) 81.5 4.04–3.98 (m) 80.4 4.01–3.94 (m) 80.5

6′′a 3.70 63.59 3.79–3.71 (m) 62.7 3.79–3.72 (m) 33.5 3.89–3.73 (m) 45.4

6′′b 3.64

J1,2

– –

4.0

–

4.0,

–

4.0

–

J2,3 9.7 10.0 9.7

J3,4 9.7 10.0 9.7

J1′a,1′b – 10.3 —

J3′,4′ 8.6 8.6 8.6

J3′′,4′′ 8.6 8.6 8.6 71

Calub, T. M.; Waterhouse, A. L.; Chatterton, N. J. Carbohydr. Res. 1990, 199, 11–17.

83

2.4 Conclusion

Since halodeoxysucrose compounds at the primary counterpart are not completely

established brief NMR assignments (See Table 3–19 in the experimental sections), this made the

difficulties to distinguish between 6- and 6′-monohalo sucrose isomers based on 1H-NMR, due to

overlapped signals of protons at 6- and 6′-positions with some other positions. These tendencies

also promoted ambiguous result for Appel reaction reactivity. In this study, comprehensive NMR

analyses for each monohalogenated sucrose moiety at the primary position is described and even

constructed the reactivity of the Appel reaction based on its isolated per-O-acetylated compounds.

The NMR characterization was then revisited for 6′-bromo-6′-deoxysucrose heptaacetate (2) to

show that structural assignment of this molecule in the previous report was misinterpreted and

needed to revise their assignment as the 6-bromo-6-deoxysucrose heptaacetate (3). Due to this

favor, the de-O-acetylated 6′-bromo-6′-deoxysucrose (14) can be purely assigned in this study.

However, the regioselective Appel reaction of sucrose followed the halogenation order of

6>6′>>1′. Additionally, chemoenzymatic installation of monohalogenated sucrose derivatives at

the 1′-position led to the first reported synthesis of compounds 1′-bromo-1′-deoxysucrose

heptaacetate (10) and 1′-bromo-1′-deoxysucrose (20). The establishment of three

monohalogenated sucrose isomers modified at the primary position will be useful to extend the

novelty and diversity of carbohydrate-based products. Moreover, further modification at these

primary positions with representative probe, such as benzophenone or [3-(trifluoromethyl)phenyl]

diazirine, can be useful for photoaffinity labeling analysis of human sweet taste receptor.

1-Kestose, one of FOS with additional fructose moiety at 1′-position of sucrose, treatments

with Appel reaction resulted in trihalogenated 1-kestose at the 6-, 6′-, and 6′′-positions (See

Table 20–21 in the experimental sections for detail NMR analysis). Isolation and structure

elucidation were easily completed using the per-O-acetylated form which the 1D and 2D

NMR supported the halogenated positions. The synthesis and structure elucidation of novel

compounds 1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-tribromo-6,6′,6′′-trideoxy-1-kestose (24)

and 1′′,2,3,3′,3′′,4,4′,4′′-octa-O-acetyl-6,6′,6′′-trichloro-6,6′,6′′-trideoxy-1-kestose (25)

contribute to the introduction of halogenations of primary hydroxy groups of FOS, which can

potentially be used as low-calorie and non-digestible sweeteners as artificial sugar for

diabetics.

84

Chapter 3 -Amino acid extensive acylation

3.1 Introduction

The widely used C-acylation, Friedel–Crafts acylation28,29

of aromatic compounds are

efficient methods that result in satisfactory product yields.34

Conventionally, the Friedel–

Crafts acylation of aromatic derivatives with acyl donor requires at least 1 equiv. of AlCl3 as

catalyst.29,35

The improvement on this reaction for various substrates, including α-amino acid,

is continuing at the time of writing this dissertation. Thus, to extend the acylation of α-amino

acid, this part is refined into two parts. The high storage stability of N-hydroxysuccinimide

ester that impressively used for direct acylation and very reactive proportion of conventional

acid chloride that still interesting to explore its ability, which both used as representative acyl

donor in this chapter.

Figure 20 N-protected -amino aryl-ketone synthesis methods33

Chiral N-protected -amino aryl-ketones are usually used as a precursor in the synthesis

of various biologically active compounds.26,32

There are several approaches for their syntheses,

which generally can be devided as ones that use α-amino acid derivatives or non-amino acid

derivatives as reactants (Figure 20).33

When non-amino acid is used as reagents, the multistep

reactions, the use of special catalyst, and the requirement for time-consuming chiral resolution

are the problems for this method. Meanwhile, utilization of α-amino acid derivatives as

reagents might offer convenient method since optically active α-amino acid can be used as

starting material. Among this method, Friedel–Crafts acylation28,29

of arenes was taken part in

N-protected -amino aryl-ketone

α-Amino acid derivatives as reactants

Friedel–Crafts acylation Condensation or cross coupling

Non-Amino acid derivatives as reactants

Chiral amine-donor reaction with phenyl ketone

85

the most favorable strategy to synthesize -amino aryl-ketone (Figure 20), which results in

satisfactory product yields.34

Figure 21 Friedel–Crafts acylation with utilization of various acyl donors to synthesize N-

protected -amino aryl-ketone. Protecting group is abbreviated as “PG”

The -amino acid chloride25,26,31,32

is widely used as an acyl donor to undergo Friedel–

Crafts acylation due to its high reactivity (Figure 21). However, this acyl halide is unstable,

sensitive to moisture and difficult to handle.83

Therefore, it should be used instantly and

cannot be stored. In some cases, utilization of -amino acid chloride also cannot prevent the

racemization.26,83

Moreover, -amino acid anhydride (Figure 21) can be employed as an

acylating agent and can be used to obtain the -amino aryl-ketone without the N-protecting

group. Unfortunately, -amino acid anhydride only shows reactivity for electron-rich arene

acyl acceptor84

and needed to be prepared by using a toxic gas, such as phosgene or

triphosgene85

Since -amino acid anhydride consists of two possible -amino acids, one of

the non-acylated molecules will be wasted during the reaction. Recently, N-protected N-(-

aminoacyl)benzotriazole (Figure 21) has been reported to acylate benzene by using Lewis

acid.30

Despite this N-acylbenzotriazole being more convenient to handle compared to -

amino acid chloride, only a moderate yield of -aminoacyl phenyl-ketone can be synthesized.

However, the preparation of N-protected N-(-aminoacyl)benzotriazole30,86,87

was conducted

under in situ conditions, which indicated that its isolation takes more effort.

Types of acyl donor

N-protected -amino acid chloride

N-protected -amino acid anhydride

N-protected -amino acid benzotriazole

86

N-Hydroxysuccinimide ester (OSu) derivatives of α-amino acids have high storage

stability88

and have enough reactivity with amino components under moderate condition.

Several hundreds of OSu modified in α-amino acid derivatives are commercially available

which widely used as convenient reagents in peptide synthesis. The OSu modified in α-amino

acid is also known to be facilitated peptide synthesis as good alkoxy leaving group.89

This

utility indicated that the OSu derivatives may act as acyl donor for Friedel–Crafts reaction. In

peptide construction via amide bond formation, it is known to produce fewer side reactions,

including racemization. Moreover, it reacts cleanly, thus suitable for purification with

partition.89

The Friedel–Crafts acylation of electron-rich arene (ferrocene and pyrene) with N-

hydroxysuccinimidyl benzoic or p-methoxybenzoic acid has been previously reported, of

which reagents were activated by superacidic trifluoromethanesulfonic acid.90

Up to date, no

study has been reported the α-amino acid-OSu as representative skeleton for direct Friedel–

Crafts acylation. In this work, the synthesis and properties of potential acyl donor for Friedel–

Crafts acylation, N-trifluoroacetyl (TFA)-protected α-amino acid-OSu, are described. TFA

protection of α-amino acid is preferred due to its stability to Lewis Acid to undergo acylation

with conventional Friedel–Crafts catalyst, AlCl3.

Until now, no study has been reported as the use of -amino acid-OSu for a

representative skeleton in direct Friedel–Crafts acylation. In this study, the synthesis and

properties of a potential acyl donor for Friedel–Crafts acylation, namely N-trifluoroacetyl

(TFA)-protected -amino acid-OSu, are described. The demonstrated utility of optically

active isoleucine, which has two chiral centers in the molecules, and its diastereomer allo-

isoleucine to identify chilarity at the -proton of the Friedel–Crafts acylation products by

nuclear magnetic resonance (NMR) is studied. The use of -amino acid-OSu derivatives is

expected to extend the synthesis of various TFA-protected -amino aryl-ketone which

assumed to retard its chirality which might act as a potential acyl donor for the Friedel–Crafts

acylation.

3.2 Result and discussion

3.2.1 Friedel–Crafts Acylation of aliphatic -amino acid derivatives: Isoleucine derivatives

synthesis and modification

Preparations of α-amino aryl-ketone via Friedel–Crafts acylation is catalyzed by Lewis

or Brønsted acid, which majorly utilized toxic, corrosive and moisture-sensitive acylating

reagents such as acyl halides and anhydrides. N-Hydroxysuccinimide ester (Figure 22) or so-

called “active ester” is readily accessible and stable compound, in which benzoic acid N-

hydroxysuccinimide esters had been reported efficiently acylated ferrocene and pyrene under

87

a strong acidic condition.90

N-hydroxysuccinimide ester might give merit as an acyl donor for

Friedel–Crafts acylation due to its exhibiting high reactivity towards the amino group and

widely used for synthesis of biomolecules by C-N bond formation (Amide bond formation,

Figure 22).89

N-Hydroxysuccinimide ester is having high reactivity and easy to purify. It

reacts cleanly and rapidly, moreover, the co-product is water-soluble (Figure 22). It also can

produce less side reactions during the coupling reaction, including racemization for amide

bond formation.89

Therefore, N-hydroxysuccinimide ester might act as promising acyl donor

for synthesis of aryl-keto α-amino acids.

Figure 22 N-Hydroxysuccinimide ester (OSu) as representative reagent for amide bond

formation

L-Isoleucine is known to have the ability to undergo epimerization at the -position to

produce D-allo-isoleucine. As for chiral N-protected -amino aryl-ketone synthesis, especially

the reaction involving direct acylation of -amino acids, the chirality of product is commonly

determined by complex formation with a chiral shift reagent.25

This racemic -amino aryl-

ketone can be detected by the appearance of two separated proton signals in 1H-NMR

spectrum. Up to now, there is no reported study that has checked the chirality of the TFA-

protected -amino aryl-ketone based on isoleucine and its diastereomer allo-isoleucine. Since

the asymmetric carbon at -position within these stereoisomers can be detected by NMR,41

utilization within isoleucine and its diastereomer allo-isoleucine (four types of stereoisomers;

Figure 4) might be useful in describing a change in configuration of their chiral center during

the chemical modification.

Traditionally, the coupling of amino acids in amide bond formation is utilizing N-

protecting group, such as urethane-based protecting group such as t-butyl (BOC), benzyl

(Cbz) or fluorenylmethyloxycarbonyl (Fmoc) that predominantly causing recemization.91

In

spite of this problem that cause by other N-protecting group, TFA-protected amino acid might

proceed chirality retention in Friedel–Crafts reaction.25,32

Therefore, initially in this study, the

corresponding amino acid of optical active L-isoleucine (L-Ile, L-31) underwent TFA

protection by using ethyl trifluoroacetate in the presence of triethylamine in methanol92,93

to

Amide bond

formation

N-Hydroxysuccinimide

Ester (OSu)

Water-soluble

co-product

88

generate TFA-L-Ile (L-32, Scheme 11). Next, the L-32 was transformed into TFA-L-Ile-OSu

(L-33) within 3 h at room temperature by utilization of 1.1 equiv. NHS (N-

hydroxysuccinimide) and 1.0 equiv. of WSCD-HCl in CH2Cl2 (Scheme 11). TFA-L-Ile-OSu

(L-33) is expected as potential acyl donor for Friedel–Crafts acylation which was described in

this study.

Scheme 11 Synthesis TFA-L-isoleucine-N-hydroxysuccinimide ester (TFA-L-Ile-OSu, L-33)

The L-33 is stable for storage at −20 °C for more than six months and can be readily

used as an acyl donor to undergo Friedel–Crafts acylation into benzene 34. The L-33 was

found to be solved in excess amount of benzene 34 (300 equiv.) which sufficient for the

purpose in this study. Preliminarily, an excess amount (6 equiv.) of conventional Friedel–

Crafts catalyst of AlCl3 was tested for the reaction at room temperature (Scheme 12 (a)). The

room temperature reaction took longer reaction time and found not only moderate proportion

of desired phenyl ketone of TFA-L-Ile-Ph (L-35) but also the starting material still remained

after 24 h. Thus, the reaction was conducted at higher temperature (70 oC, Scheme 12 (b)),

and within 2 h, the reaction is completed to result in L-35 with a fine yield.

Scheme 12 Friedel–Craft acylation of benzene (34) with TFA-L-Ile-OSu (L-33), in which

utilizing conventional catalyst of AlCl3 was utilized at (a) room temperature and (b) 70 oC.

a

Calculated from consumption of TFA-L-Ile-OSu (L-33).

89

Table 22 Friedel–Craft acylation of benzene 34 with TFA-L-Ile-OSu (L-33) using various

Lewis acids as catalyst.

Entry Lewis acid Equiv. Time L-35

(% Yield)a

1 AlCl3 1.5 3 d N.R.b

2 AlCl3 3 9 h N.R.c

3 AlCl3 6 2 h 86

4 SnCl2 6 7 d ≤ N.R.e

5 ZnCl2 6 7 d ≤ N.R.e

6 FeCl3 6 2 d N.R.e

7 TiCl4 6 1 d N.R.e

8 GaCl3 6 2 h 4c,f

9 InCl3 6 2 h N.R.b

a N.R. stands for no reaction. b Starting material of TFA-L-Ile-OSu (L-

33) remained. c A complex mixture was recovered. e Hydrolysis is

preferred rather than acylation. TFA-L-Ile (L-32) was recovered. f

Calculated from 1H-NMR.

The success of previous utilization of Friedel–Crafts acylation at 70 oC for 2 h can

establish desired product of L-35 with a fine yield, then by the same temperature, lower

amounts of AlCl3 are also tested. When 1.5 equiv. of AlCl3 was used (Table 22 Entry 1), the

small proportion of AlCl3 was not enough to conduct the reaction and starting material L-33

still remained. The effort to increase the AlCl3 proportion up to 3 equiv. (Table 22 Entry 2)

resulted in complicated mixture. Therefore, utilization of 6 equiv. of AlCl3 is believed to

effective for this reaction (Table 22 Entry 3).

The screening of Lewis acid utilization for acylation of L-33 to benzene has been

examined. When the conventional catalyst AlCl3 (Table 22, Entry 3) was replaced with other

common Lewis acids (Table 22, Entries 4–7) under the same reaction conditions (6 equiv.

Lewis acid, at 70 °C), instead of desired product formation, hydrolysis occurred and L-35 was

detected after quenching the reaction mixture. Another metal halide, GaCl3, contributed to the

90

acylation, which resulted in low yield of L-35 (Table 22, Entry 8). Compared with GaCl3,

InCl3 showed the lowest reactivity due to remaining the starting material of L-33 recovered

after the reaction mixture was quenched (Table 22, Entry 9). However, the L-35 was produced

from acylation utilized by GaCl3 and showed retention of -proton chirality, the chemical

yield of the desired -amino phenyl-ketone is far less than that produced with the reaction

utilizing AlCl3. If an excess AlCl3 was used, the Lewis acid apparently will coordinate to the

most basic site94

(the carbonyl oxygen atoms of OSu and N-acyl groups) and Friedel–Crafts

acylation can take place, resulting in a high yield of the desired product. Therefore, AlCl3 is

preferred to undergo the reaction due to its high reactivity and also the fact that it has a

considerably lower cost that brings economic benefits.

L-Isoleucine (L-Ile, L-31) achived for modification into active ester of OSu and its

application for synthesis of -amino phenyl ketone via Friedel–Crafts acylation led this study

to extent the other isoleucine derivatives utilization. First, D-isoleucine (D-Ile, D-31) followed

the similar modification with L-31. The optical active D-31 underwent TFA protection to

generate TFA-D-Ile (D-32, Scheme 13 (a)). The resulted D-32 then modified at its C-terminal

with OSu to form TFA-D-Ile-OSu (D-33) and utilized it for acylation into benzene 34 which

produced phenyl ketone of TFA-L-Ile-Ph (L-35) with a fine yield.

The use of racemization reaction of L-isoleucine into D-allo-isoleucine is beneficial in

geochronology.40

The racemization rate for these derivatives is relatively slow which suitable

for utilization in aging prediction methods. This chemical feature of diastereomer of

isoleucine derivative, the optical active L-/D-allo-isoleucine (L-/D-allo-Ile, L-/D-36) can also

be employed for checking the chirality retention of -amino acids during its modification.

Hence, L-/D-allo-Ile (L-/D-36) is undergone the similar N-terminal protection methods of

other isoleucine derivatives to result in TFA-L-/D-allo-Ile (L-/D-37, Scheme 13 (b) and (c)).

By the same manner, TFA-L-/D-allo-Ile (L-/D-37) was modified into TFA-L-/D-allo-Ile-OSu

(L-/D-38) which was then utilized as representative acyl donor to result in TFA-L-/D-allo-Ile-

Ph (L-/D-39, Scheme 13 (b) and (c)).

91

Scheme 13 Synthesis and application of (a) TFA-D-isoleucine-N-hydroxysuccinimide ester

(TFA-D-Ile-OSu, D-33); (b) TFA-L-allo-isoleucine-N-hydroxysuccinimide ester (TFA-L-allo-

Ile-OSu, D-38); (c) TFA-D-allo-isoleucine-N-hydroxysuccinimide ester (TFA-D-allo-Ile-OSu,

D-38).

92

Figure 23 Selected 1H-NMR (270 MHz, CDCl3) of (a) TFA-L-/D-Ile (L-/D-32) and TFA-L-/D-allo-Ile (L-/D-37); (b) TFA-L-/D-Ile-OSu (L-/D-33) and

TFA-L-/D-allo-Ile-OSu (L-/D-38); (c) TFA-L-/D-Ile-Ph (L-/D-35) and TFA-L-/D-allo-Ile-ph (L-/D-39)

TFA-L-Ile-OSu (L-33)

TFA-D-Ile-OSu (D-33)

TFA-L-allo-Ile-OSu (L-38)

TFA-D-allo-Ile-OSu (D-38)

5.3 5.0 4.7

δ 1H (ppm)

TFA-L-Ile (L-32)

TFA-D-Ile (D-32)

TFA-L-allo-Ile (L-37)

TFA-D-allo-Ile(D-37)

5.0 4.7 4.4

δ 1H (ppm)

Hα

Hα

TFA-L-Ile-Ph (L-35)

TFA-D-Ile-Ph (D-35)

TFA-L-allo-Ile-Ph (L-39)

TFA-D-allo-Ile-Ph (D-39)

6.0 5.7 5.4

δ 1H (ppm)

Hα

Hα

Hα

Hα

(a) (b) (c)

93

In Figure 23, the asymmetric carbon at -position within stereoisomers of isoleucine

and its diastereomer allo-isoleucine can be detected as distinguishable methine proton signal

by 1H-NMR. The optically active L-/D-32 and L-/D-37 show nonequivalence signals arising

from epidemically related -proton (Figure 23 (a)). The comparison between L-/D-32 (δH 4.68

ppm) and its diastereomer L-/D-37 (δH 4.76 ppm) shows differences of the chemical shift at

the -proton appearing in 1H-NMR spectra, which suggested that isoleucine derivatives are

sufficient tools for checking the epimerization during the reaction. The characterization of

isoleucine derivatives activated at C-terminal by OSu (L-/D-33 and L-/D-38) by 1H-NMR can

serve the purpose for checking the chirality preservation41

during the modification or reaction

process, in which only the -proton signal of L-/D-33 in 1H-NMR (δH 4.97 ppm, Figure 23

(b)) can be observed for identification of isoleucine derivatives and no trace of L-/D-38 (δH

5.10 ppm) was detected (Figure 23 (b)).

Based on 1H-NMR (Figure 23 (c)), the chiral center at -position of TFA--amino-

phenyl ketone derivatives L-/D-35 that were synthesized via Friedel–Crafts acylation of TFA-

-amino-OSu derivatives with benzene 34 clearly shows no appearance of any

nonequivalence signals except for that at δH 5.60 ppm. Meanwhile, proton signal at α-methine

of L-/D-39 should be detected at δH 5.74 ppm. This indicates that either L-/D-1c or its

diastereomer, different in chiral center only at the -position of L-/D-39, shows no

epimerization after the acylation. Compared with the acylation of aryl N-hydroxysuccinimide

ester derivatives and electron-rich arenes, such as ferrocene and pyrene,90

L-/D-33 and L-/D-38

are reactive in relatively electron-poor acyl acceptors when conventional AlCl3 is used. Thus,

the activation is not necessary under harsh conditions, such as utilization of super acidic

trifluoromethanesulfonic acid.33,34

Moreover, L-/D-33 and L-/D-38 can dissolve well in

benzene and thus, no solvent is needed for the reaction system. In comparison, the reported N-

protected N-(-aminoacyl)benzotriazole, which was previously used as an acyl donor for the

Friedel–Crafts reaction, showed low solubility for the selected acyl acceptor and utilized

CH2Cl2 as the solvent.30

However, when isoleucine derivative and its diastereomer allo-isoleucine derivative are

utilized for checking the chiral center by 1H-NMR (Figure 24), it can offer a simple analysis

that is able to comprehensively examine all the processes of TFA-protected -amino acid-

OSu synthesis as the acyl donor of Friedel–Crafts acylation. To support our understanding,

the optical rotation of all corresponding L-/D--amino acid derivatives was also conducted for

checking the retention given as -proton chirality during the process. Moreover, based on our

finding of -protons in 1H-NMR (Figure 23), it appears that the formation of acylium cation

94

(Figure 3 (b)), given as the common Friedel–Crafts intermediate, can successfully retard the

-proton chirality.

Figure 24 Correlation of isoleucine derivative and its diastereomer allo-isoleucine derivative

for checking the chiral center by 1H-NMR

For further applications of -amino acid-OSu as a potential acyl donor that can be easily

activated by the conventional Friedel–Craft catalyst of AlCl3, L-33 was then used in acylation

reactions to convert it with various arenes (Table 23). The L-33 reacted with 300 equiv. of

arenes (40–44) by using the same condition with the previously optimized condition for

acylation of benzene (34) which is utilized of 6 equiv. of AlCl3 and reacted for 3 h. TFA-L-

Ile-Ar (L-45, L-47, L-49–L-51) was the main isolate with a fine yield. The acylation into

electron-donating arenes, such as toluene 40 and anisole 41 mainly occurs at a less hindered

position (p-position is prior to o-position with ratio L-45:L-46 is 5:1 and L-47:L-48 is 18:1,

respectively). From 1H-NMR, only the -proton signal of desired L-45, L-47, and L-49–L-51

can be observed which implies that there is no other diastereomers showing the -proton

chirality retention of TFA-protected -amino aryl-ketone.

95

Table 23 Friedel–Crafts reaction of TFA-L-Ile-OSu (L-33) into various arenes (40–44) that

were catalyzed by AlCl3 to yield TFA-L-Ile-Ar (L-45–L-51)

Entry Arene TFA-L-Ile-Ar %Yield

1 40

TFA-L-Ile-Ph(2-Me) (L-46)

(5:1)

71

2

41

TFA-L-Ile-Ph(2-OMe) (L-48)

(18:1)

72

3 42

78

4

43

78

5

44

79

96

Scheme 14 Typical reaction of acylation by Weinreb amides system

Unlike TFA-protected -amino acid-OSu that can utilize various commercially

available acyl acceptors, Weinreb amides (N-methoxy-N-methylamides,27

popular methods

for the synthesis of -amino aryl-ketones,95

Scheme 14) are considerably less efficient for

direct acylation due to limitation of Grignard or organolithium reagents, which sometimes

need to be synthesized before use. In this study, the urge for choosing TFA-protecting group

is due to its stability in the presence of Lewis acid, which is an essential reagent for

conventional Friedel–Crafts acylation. Moreover, because of extremely basic condition of the

Weinreb amides system, only carbamate-based protecting group such as t-butyl (BOC),

benzyl (Cbz) or fluorenylmethyloxycarbonyl (Fmoc), that stable under alkaline condition, is

utilized. The limited study of previous TFA-protected -amino aryl-ketone synthesis during

the last decade is due to this reason. Therefore, the introduction of TFA--amino-OSu played

an important role as representative acyl donor for synthesis of new compound. For example,

TFA-D-Ile-OSu (D-33) and TFA-L-/D-allo-Ile-OSu (L-/D-38) are considered as novel acyl

donor for Friedel–Crafts acylation. The resulted amino phenyl-ketone of TFA-L-/D-Ile-Ph (D-

35) and TFA-L-/D-allo-Ile-Ph (L-/D-39) are also novel compounds that contribute in the study

of TFA-protected -amino aryl-ketone synthesis.

3.2.2 Friedel–Crafts Acylation of aliphatic -amino acid derivatives: Synthesis and

modification of various aliphatic -amino acid derivatives

Friedel–Crafts acylation for synthesis of aryl-keto α-amino acids is commonly utilized

α-amino acid chloride25,32

which is known for its high reactivity for this reaction. Katritzky et

al.30,86

also reported the used of α-aminoacyl benzotriazole as an amide-type acyl donor for

comprehensive aryl-keto α-amino acid synthesis without any optical loss. However, ester type

of acyl donor utilized in α-amino acids is still far to explore.

In 2000, Olah and co-workers96

introduced the benzoic acid methyl ester as a novel acyl

donor to undergo Friedel–Crafts acylation. Even by using the highly deactivated nitrobenzene

and benzotrifluoride as acyl acceptor, acylation with benzoic acid methyl ester catalyzed by

strong acid still showed good reactivity. Followed by this ester utilization, the benzoic acid N-

97

hydroxysuccinimide ester, benzoic acid tetrafluorophenyl ester, and benzoic acid phenyl ester

are reported to be effective for acylation of ferrocene and pyrene under strong acid conditions.

However, the contribution of ester as an acyl donor is broadening selection of active skeleton

for more comprehensive acylation. In this part, potential acyl donor of TFA-α-amino acid-

OSu is also tried for other aliphatic α-amino acids as described below.

Synthesis and application of TFA-glycine-N-hydroxysuccinimide ester

Scheme 15 Synthesis of TFA-Gly (52)

Glycine (Gly, 51) is the simplest natural α-amino acid that does not contain chiral center.

It is an important -amino acid the formation of porphyrins (skeleton of metal-containing

oxygen-transport protein in red blood cell (eukaryotes)) which can undergo the condensation

with succinyl-coenzyme A (CoA) from citric acid cycle to yield -amino-aryl ketone.97

Since

glycine ketone is substantial in eukaryotes, glycine was also tried for Friedel–Crafts acylation.

First, glycine (51) was underwent TFA protection to obtain TFA-Gly (52) in a satisfied yield

(Scheme 15).

Scheme 16 Synthesis of TFA-Gly-OSu (53). a

WSCD-HCl is directly added into reaction.

Solvent was removed under reduced pressure. Then, residue was dissolved in ethyl acetate

and washed by 1M HCl.

98

Next, TFA-Gly (52) was directly transformed to N-hydroxysuccinimide ester by of 1.0

equiv. of WSCD-HCl and 1.1 equiv. of NHS to yield TFA-Gly-OSu (53, Scheme 16).

Utilization of dichloromethane (Scheme 16 (a)) was less effective as a solvent for this

reaction, and then the solvent was changed to dimethylformamide that resulted in TFA-Gly-

OSu (53) with an excellent yield (Scheme 16 (b)). The resultant 53 was then directly tested

for Friedel–Crafts acylation by reaction with benzene at 70 oC to produce TFA-Gly-Ph (54,

Scheme 17). In line with previous utilization of isoleucine derivatives, acylation of TFA-Gly-

OSu (53) at 70 oC was sufficient to giving TFA-Gly-Ph (54) with a fine yield.

Scheme 17 Application of TFA-Gly-OSu (53) to result in TFA-Gly-Ph (54)

Synthesis and application of TFA-L-/D-alanine-N-hydroxysuccinimide esters

Scheme 18 Synthesis of TFA-L-/D-Ala (L-/D-56)

As natural α-amino acid that contains a methyl group side chain in comparison to

glycine 51, optically pure L-/D-alanine (L-/D-Ala, L-/D-55) is the most primary amino acid

released by muscle that is converted to glucose by liver for maintaining sugar level in blood

of fasted man.98

In this study, the important -amino acid of L-/D-Ala (L-/D-55) was directly

N-terminal protected by TFA protection to result in TFA-L-/D-Ala (L-/D-56, Scheme 18).

Ester formation for carbonyl activation of TFA-L-/D-Ala (L-/D-56) was conducted by

utilization of WSCD-HCl and NHS to result in TFA-L-/D-Ala-OSu (L-/D-57, Table 24).

99

Table 24 Optimization of synthesis of TFA-L-/D-Ala-OSu (L-/D-57)

Entry Material

56

WSCD-HCl

(equiv.)

NHS

(equiv.) Solvent

57

(%Yield)

1 L- 1.3a 1.3 DMF 60

b

2 D- 1.3a 1.3 DMF 53

b

3 L- 1.3a 1.1 Acetone 53

b

4 D- 1.3a 1.1 Acetone 52

b

5 L- 1.3a 1.1 CH2Cl2 51

b

6 D- 1.3a 1.1 CH2Cl2 56

b

7 L- 1.0 1.1 CH2Cl2 52

8 D- 1.0 1.1 CH2Cl2 42

9 L- 1.0a 1.1 CH2Cl2 75

c

10 D- 1.0a 1.1 CH2Cl2 71

c

a WSCD-HCl is directly added to reaction mixture. b Solvent was removed under

reduced pressure. Then, residue was dissolve in ethyl acetate and washed by H2O,

1M HCl, sat. NaHCO3, and sat. NaCl. c Reaction mixture is directly washed by sat.

NaCl.

Since TFA-Gly-OSu (53, Scheme 16) can be successfully synthesized by utilization of

dimethylformamide, the reaction of L-/D-56 with 1.3 equiv. WSCD-HCl and 1.3 equiv. NHS

was conducted in dimethylformamide used as the solvent. However, only moderate yield of L-

/D-57 was produced (Table 24, Entries 1–2). The attempt to increase the yield of L-/D-57 was

continued by replacing the solvent with acetone and dichloromethane (Table 24, Entries 2–6),

but no improvement was found. The harsh condition might take place in the first place; thus

the reagent proportion was lowered (Table 24, Entries 2–6). Since the yield of L-/D-57 was not

improved after the tested condition, the consideration for the purification method by washing

directly with sat. NaCl was performed, which can increase the fine yield of L-/D-57 (Table 24,

Entries 2–6). Thus, the representative L-/D-57 can be readily used as acyl donor for Friedel–

Crafts acylations.

100

Scheme 19 Application of TFA-L-/D-Ala-OSu (L-/D-57) to result in TFA-L-/D-Ala-Ph (L-/D-

58) at (a) 70 oC and (b) room temperature

Conventional Friedel–Craft by utilization of AlCl3 is applied for acylation of L-/D-57

with benzene to produce TFA-L-/D-Ala-Ph (L-/D-58) at 70 oC for 2 h (Scheme 19 (a)). In the

previous study, TFA N-(α-aminoacetyl)benzotriazole is reported to acylate benzene by the use

of AlCl3 at 20 oC for 3 h and resulted in α-aminoacetyl phenyl-ketone in the moderate yield

(63% yield).30

For comparison, L-/D-57 also tested at room temperature for 36 h which

resulted in fine yield of L-/D-58 (78–84% yield, Scheme 19 (b)). This result ensured L-/D-58

reactivity as potential acyl donor for this reaction and its high solubility under excess of

benzene is sufficient for resulting high yield of ketone product. In similar with isoleucine

derivatives utilization, acylation by the used L-/D-57 is efficient and resulted in L-/D-58

without loss of chirality.

Synthesis and application of TFA-L-/D-valine-N-hydroxysuccinimide esters

After the success of simple -amino acids, such as glycine (51) and L-/D-alanine (L-/D-

55), for synthesis of -amino phenyl ketones via N-hydroxysuccinimide ester formation, a

branched-chain of natural α-amino acid, such as L-/D-valine (L-/D-59), was also tested whether

it is accepted as a potential acyl donor after conversion to the TFA-α-amino acid-OSu. First,

optically pure L-/D-valine were protected by TFA protection and resulted in TFA-L-/D-valine

(TFA-L-/D-Val, L-/D-60) (Scheme 20). Next, L-/D-60 were transformed to TFA-L-/D-Val-OSu

(L-/D-61) within fine yield (Scheme 20). The TFA-L-/D-Val-Ph (L-/D-62) were produced

under conventional Friedel–Crafts condition after the reaction of L-/D-61 with benzene for 4 h

101

(Scheme 20). Organic synthesis and modification that based on L-/D-valine (L-/D-59) chemical

feature in this study allowing the synthesis of novel compounds, TFA-D-Val-OSu (D-60) and

TFA-D-Val-Ph (D-61), which contribute into vast exploration for peptide synthesis in the

future.

Scheme 20 Synthesis and application of TFA-L-/D-Val-OSu (L-/D-61)

Synthesis and application of TFA-L-/D-leucine-N-hydroxysuccinimide esters

The other typical branched-chain of natural α-amino acid besides L-/D-valine (L-/D-59)

is L-/D-leucine (L-/D-63) which also underwent modification to become potential acyl donor

of α-amino acid-OSu. The optically pure L-/D-63 were protected by TFA protection and

resulted in TFA-L-/D-Leu (L-/D-64, Scheme 20), respectively. Next, by the same condition

when utilization of isoleucine derivatives, TFA-L-/D-Leu (L-/D-64) were transformed to TFA-

L-/D-Leu-OSu (L-/D-65, Scheme 20) within fine yield.

The TFA-L-/D-Leu-Ph (L-/D-66) can be produced under a conventional Friedel–Crafts

condition after the reaction of L-/D-65 with benzene at 70 oC for 2 h (Scheme 21). The TFA-

D-Leu-OSu (D-65) is considered as a novel compound which was synthesized in this study.

Moreover, due to stability of TFA-L-/D-Leu-OSu (L-/D-65) for Friedel–Crafts acylation, the

novel compound of TFA-L-/D-Leu-Ph (L-/D-66) can be synthesized successfully. Based on the

comparison between optical acitive alanine, valine, or leucine, utilization of N-

hydroxysuccinimide ester is independent with branched-chain of -amino acids in which

102

reaction of TFA-α-amino-OSu can result in fine yield of -amino phenyl ketone without loss

of chirality.

Scheme 21 Synthesis and application of TFA-L-/D-Leu-OSu (L-/D-65). a Calculated from

1H-

NMR.

Synthesis and application of TFA-L-/D-norvaline-N-hydroxysuccinimide esters

Comprehensively, the none-branched-chain of unnatural aliphatic α-amino acid of

optically pure L-/D-norvaline (L-/D-Nva, L-/D-67) is utilized in this study. After completion the

TFA protection, the resultant TFA-L-/D-Nva (L-/D-68, Scheme 22) were directly transformed

to TFA-L-/D-Nva-OSu (L-/D-69) using WSCD-HCl and NHS (Scheme 23). Within the same

equiv. of NHS (1.1 equiv.) for this typical reaction, slightly higher proportion of WSCD-HCl

from 1.0 equiv. (Scheme 23 (a)) into 1.1 equiv. (Scheme 23 (b)) was utilized, and TFA-L-/D-

Nva-OSu (L-/D-69) formation can be improved. The reaction was conducted at 0 oC due to

exothermic reaction when L-/D-67 were used.

Scheme 22 Synthesis of TFA-L-/D-Nva (L-/D-68)

103

Scheme 23 Synthesis of TFA-L-/D-Nva-OSu (L-/D-69) by utilizing (a) 1.0 equiv. of WSCD-

HCl and (b) 1.1 equiv. of WSCD-HCl.

The representative acyl donors of L-/D-69 also showed high reactivity toward benzene

34 at 70 oC; that resulted in TFA-L-/D-Nva-Ph (L-/D-70) with a fine yield by 3 h (Scheme 24

(a)). Similar with natural -amino acids that were previously tested for the synthesis of -

amino phenyl ketone derivatives, L-/D-69 showed high reactivity toward benzene and L-/D-70

can be formed at room temperature (Scheme 24 (b)). Although N-protected -amino-OSu

derivatives is known for its stability and reactivity in peptide synthesis,89

its application for

unnatural -amino acids has still not been yet fully explored. Thus, the synthesized novel

compound L-/D-69 which can be utilized for the synthesis of L-/D-70 can contribute to

synthesis of new compounds and broaden exploitation for modification of the -amino acid

structures.

Scheme 24 Application of TFA-L-/D-Nva-OSu (L-/D-69) to result in TFA-L-/D-Nva-Ph (L-/D-

70) at (a) 70 oC and (b) room temperature.

aCalculated from

1H-NMR.

104

Synthesis and application of TFA-L-/D-norleucine-N-hydroxysuccinimide esters

To expand the application of unnatural alphatic α-amino acid exploration, the longer

none-branched-side chain of L-/D-norleucine (L-/D-Nle, L-/D-71) was tested in this study. N-

Terminal of L-/D-71 was protected with TFA to give TFA-L-/D-Nle (L-/D-72, Scheme 25).

When TFA-L-/D-Nle (L-/D-72) subjected with WSCD-HCl and NHS for synthesis of TFA-L-

/D-Nle-OSu (L-/D-73), there is no improvement for the product yield after reaction with

different WSCD-HCl proportion (Scheme 26). Utilization of 1.0 equiv. of WSCD-HCl

(Scheme 26 (a)) was found to produce not only L-/D-73 due to observation of unidentified

compound by 1H-NMR, while utilization of 1.1 of equiv. WSCD-HCl (Scheme 26 (b)) was

preferred to conduct this reaction. The moderate yield of L-/D-73 was sufficient for further

application as acyl donor for Friedel–Crafts acylation.

Scheme 25 Synthesis of TFA-L-/D-Nle (L-/D-72)

Scheme 26 Synthesis of TFA-L-/D-Nle-OSu (L-/D-73) by utilizing (a) 1.0 equiv. of WSCD-

HCl and (b) 1.1 equiv. of WSCD-HCl. a Contaminated with unidentified compound, observed

as another α-proton signal in 1H-NMR spectrum (ratio 1.00:0.06).

105

The longer aliphatic side-chain of L-/D-71, compared with L-/D-67, is not hampered its

reactivity for the reaction with benzene to result in TFA-L-/D-Nva-Ph (L-/D-74). The L-/D-73

showed high reactivity for both reactions at 70 oC (Scheme 27 (a)) and at room temperature

(Scheme 27 (b)). These results indicate that yield of -amino phenyl ketone without loss of

chirality between optical active natural -amino acids of alanine, valine, or leucine, and

unnatural -amino acids of norvaline and norleucine utilized for N-hydroxysuccinimide ester

is independent with branched-chain or none-branched-chain of -amino acids. Moreover, the

resulted TFA-L-/D-Nle-OSu (L-/D-73) and TFA-L-/D-Nle-Ph (L-/D-74) are novel compound

which can be use for further application in bimolecular modification.

Scheme 27 Application of TFA-L-/D-Nle-OSu (L-/D-73) to result in TFA-L-/D-Nle-Ph (L-/D-

74) at (a) 70 oC and (b) room temperature.

a Calculated from

1H-NMR.

In this study, the introduction of two pure enantiomers of L- and D-α-amino acids that

were modified into TFA-α-amino acid-OSu and their utilization as potential acyl donor to

result in α-amino aryl-ketone synthesis showed identical optical rotation with opposite sign.

Hence, either TFA-α-amino acid-OSu construction or its application for acylation under

Friedel–Craft condition show the chirality retention, in which line the utilization of L-/D-

isoleucine and its diastereomer L-/D-allo-isoleucine for detection of α-amino aryl-ketone

maintained optical activity confirmable by the appearance of nonequivalence α-proton in 1H-

NMR spectra.

106

Synthesis and application of TFA-L-/D-proline-N-hydroxysuccinimide esters

The L-/D-proline (L-/D-75) are the only aliphatic α-amino acids of which secondary

amino group, or somethimes name as an imino acid, attachs to a five-member ring in a

molecule. Thus, proline is potentially tested for α-amino acid aryl-ketone synthesis. As the

substrate, L-/D-75 first was N-TFA-protected to give TFA-L-/D-Proline (L-/D-76, Scheme 28),

before transforming into TFA-L-/D-Pro-OSu (L-/D-77) within 3 h at room temperature by

utilization of 1.1 equiv. NHS and 1 equiv. of WSCD-HCl in CH2Cl2 (Scheme 28). Direct

acylation of TFA-L-/D-Pro-OSu (L-/D-77) with 300 equiv. of benzene which was activated by

6 equiv. AlCl3 can generate TFA-L-/D- Pro-Ph (L-/D-78)25,32,99

with an excellent yield (81–

84%, Scheme 28).

Scheme 28 Synthesis and utilization of TFA-L-/D-Pro-OSu (L-/D-77)

The observation of another -proton signal of proline derivatives (L-/D-76–L-/D-78) by

1H-NMR analysis sugested the existance of cis- and trans-isomers. Isomerization of N-

protected proline is known and plays a key role in the rate-determining steps of protein

folding.100

To ensure these results, 1H-NMR comparison between TFA-L-Pro (L-76) with

commercially available N-acetylated-L-Pro (Ac-L-Pro, L-79) is described in Figure 25. The

optically pure L-79 also shown two proportions of -protons signals in 1H-NMR. Hence, it

also suggested that all N-TFA-proline derivatives (L-/D-76–L-/D-78) exhibit no loss of

chirallity.

107

Figure 25 Comparison of TFA-L-Pro (L-76) with Ac-L-Pro (L-79) in 1H-NMR spectra

3.2.3 Friedel–Crafts acylation of aromatic α-amino acid derivatives: Synthesis and

modification of phenylalanine and tyrosine derivatives synthesis and modification

In recent, there is less attention to the study of N-TFA non-aliphatic α-amino acid

ketones. This might be due to difficulties in the reactive precursor synthesis. N-Protected

phenylalanine acid chloride101

previously synthesized was known to be prolonged to

racemization in peptide synthesis.91

The utilization of azlactones in phenylalanine is also

known for rapid racemization under a strong acidic condition.101

Anhydro-N-carboxy-

phenylalanine84

was previously reported to be prepared by using a toxic gas of phosgene85

and

is considerably less effective due to the existence of two possible acyl donors one of which

will be wasted after the reaction. The current N-protected phenylalanine benzotriazole30

is

reported to be more convenient to handle compared to α-amino acid chloride, but the

preparation was conducted under in situ conditions that indicate the isolation takes more effort.

In the previous part of this chapter, N-hydroxysuccinimide ester (OSu) of various

aliphatic -amino acids showed advantageous properties for direct acylation by conventional

Friedel–Crafts acylation.102

In this study, the first utilization and modification of aromatic α-

amino acid, such as phenylalanine and tyrosine, is then tried as a potential acyl donor for

acylation. Direct acylation via an active intermediate of OSu is also expected to have a high

reactivity for acylation and a high availability for storage.

108

Synthesis and application of TFA-L-/D-phenylalanine-N-hydroxysuccinimide esters

Scheme 29 Synthesis of TFA-L-/D-Phe-OSu (L-/D-82)

Initially, the optically active L-/D-phenylalanine (L-/D-Phe, L-/D-80) underwent N-TFA

protection by using ethyl trifluoroacetate in the presence of triethylamine in methanol to

generate TFA-L-/D-Phe (L-/D-81, Scheme 29). The L-/D-81 was then transformed into TFA-L-

/D-Phe-OSu (L-/D-82) within 3 h at room temperature by utilization of 1.1 equiv. NHS and 1

equiv. of WSCD-HCl in CH2Cl2 (Scheme 29). The resulting TFA-L-/D-Phe-OSu (L-/D-82)

was ready to use for acylation and could be stored at –20 oC for more than 3 months.

The corresponding TFA-L-/D-Phe-OSu (L-/D-82) were tested for acylation to benzene 34

by activation with a conventional Friedel–Crafts catalyst of AlCl3 (Table 25). At 70oC,

increasing the proportion of AlCl3 (Table 25, Entries 1–4 and 7–8) or increasing the

proportion of benzene (Table 25, Entries 5–6) resulted in a yield of desired inter-molecular

product TFA-L-/D-Phe-Ph (L-/D-83) without improvement. Similarly, with lowering the

temperature from 70 oC to room temperature (Table 25, Entries 3–4 and 13–14), no

improvement can be achieved. This phenomenone was caused by the formation of an intra-

molecular cyclization product of TFA-L-/D-cPhe (L-/D-84)30

in moderate yield (Table 25).

Comparison of TFA- L-Phe-Ph (L-83) and TFA-L-cPhe (L-84) in 1H-NMR spectra are shown

in Figure 26. From this 1H-NMR spectra, it clearly indicates aromatic signals in down field

(δH 6.8–8.2 ppm) of inter-molecular product containing around twice aromatic proton

proportion than intra-molecular product (δH 7.2–8.0 ppm). From the integration of 1H-NMR

spectra, it can be concluded that in inter-molecular product the addition of benzene after

acylation rection can be indentified. Based on this 1H-NMR observation, formation of TFA-L-

cPhe (L-84) can be clarified. The competing intra-molecular reaction specifically occurred on

aromatic α-amino acid,30,31

which presumably suggests that the generation of benzyl

carbonium ion101

is faster to react with an aromatic side chain of phenylalanine rather than

into benzene. As this phenomenon can be observed by subjection of L-/D-82 at room

temperature and by increasing the proportion of benzene (Table 25, Entries 11–16) under a

high amount of AlCl3, intra-molecular cyclization of L-/D-84 still cannot be suppressed.

109

Table 25 Friedel–Crafts reaction of TFA-L-/D-Phe-OSu (L-/D-82) into benzene 34 catalyzed

by AlCl3a

Entrya

Material

82 Tempt.

AlCl3

(equiv.)

Benzene

(equiv.) Time

Inter-molecular

product

TFA-L-/D-Phe-Ph

(L-/D-83, %Yield)

Intra-molecular

product

TFA-L-/D-cPhe

(L-/D-84, %Yield)

1 L- 70 oC 3 300 45 m 9 45

2 D- 70 oC 3 300 45 m 8 44

3 L- 70 oC 6 300 45 m 38 50

4 D- 70 oC 6 300 45 m 43 52

5 L- 70 oC 6 600 45 m 31 58

6 D- 70 oC 6 600 45 m 34 61

7 L- 70 oC 12 300 45 m 26 55

8 D- 70 oC 12 300 45 m 33 50

9 L- 70 oC 6 –

b 45 m – 64

10 D- 70 oC 6 –

b 45 m – 69

11 L- rt 12 150 2 d 40 59

12 D- rt 12 150 2 d 37 61

13 L- rt 12 300 2 d 46 43

14 D- rt 12 300 2 d 42 40

15 L- rt 12 600 2 d 40 54

16 D- rt 12 600 2 d 41 57

17 L- rt 12 –b 2 d – 69

18 D- rt 12 –b 2 d – 79

a Proportion was determined by

1H NMR in acetone-d6.

b Utilization with 300 equiv.

CH2Cl2 under reflux condition

110

Figure 26 Selected 1H-NMR comparison of TFA-L-/D-Phe-Ph (L-/D-83) with TFA-L-/D-cPhe

(L-/D-84)

The typical phenylalanine as acyl donor reaction with CH2Cl2 in the absence of benzene

that results in intra-molecular acylation is known well30,85,101

and investigation of TFA-L-/D-

Phe-OSu (L-/D-82) under this condition at 70oC (Table 25, Entries 9–10) and at room

temperature (Table 25, Entries 17–18) showed similar ration of intra-molecular cyclization

formation. Both the inter- and intra-molecular product is known to preserve the chirality

supported by identical optical rotation with the opposite sign. Based on Table 25, utilization

of TFA-L-/D-Phe-OSu (L-/D-82) showed relatively high reactivity toward acylation into

benzene under activation by AlCl3 to result in N-TFA α-amino-aryl ketones. In the contrary,

anhydro-N-carboxy-DL-phenylalanine,85

as previously reported, did not result in any α-amino-

aryl ketones.

2 x Ar-H

Ar-H

CHNH CH2CH

CHNH CH2CH

1H-NMR (270MHz,CDCl3):

1H-NMR (270MHz, Acetone-d6):

δ 1H/ ppm

δ 1H/ ppm

Intermolecular acylation

Intramolecular acylation

Inter-molecular acylation

Intra-molecular acylation

111

Since utilization with more excess benzene showed TFA-L-/D-cPhe (L-/D-84)

considerably favored formation, TFA-L-/D-Phe-OSu (L-/D-82) was also tested to various

arenes. The treatment of TFA-L-/D-Phe-OSu (L-/D-82) with 300 equiv. of benzene and 6 equiv.

of AlCl3 (Table 25, Entries 3–4) showed relatively efficient acylation, thus this condition was

utilized for acylation into various arenes. Introduction of the reaction between TFA-L-/D-Phe-

OSu (L-/D-82) and toluene 40 (Table 26, Entries 1–2) shows inter-molecular reaction as

preferred one, which resulted in TFA-L-/D-Phe-Ph(4-Me) (L-/D-85) in much moderate yield

(52–58%), rather than reaction between TFA-L-/D-Phe-OSu (L-/D-82) and benzene (Table 25).

The methyl substituent acts as an electron donating group in aromatic moiety of toluene

which might suppress intra-molecular formation of the acylated product. The occurrence of

TFA-L-/D-Phe-Ph(4-OMe) (L-/D-86) were relatively in high yield (66–68%, Table 26, Entries

3–4), supporting the effect of the electron donating group that activated aromatic moiety for a

fast electrophilic aromatic substitution during a conventional Friedel–Crafts acylation. The

methoxy group of anisole might play an important role for the suppression of intra-molecular

cyclization since no TFA-L-/D-cPhe (L-/D-84) was detected by 1H-NMR. The acylation mostly

occurs at a less hindered position (p-position) of toluene and anisole (ratio between L-/D-85

and L-/D-87 is 17:1 and L-/D-86 and L-/D-88 is 13:1, respectively). Regarding xylenes (Table

26 Entries 5–10), each of the di-methyl group substituent might induce inter- and intra-

molecular reactions. The high reactivity was shown in o-xylene that resulted in approximately

70% yield of TFA-L-/D-Phe-Ph (3,4-Me) (L-/D-89) which suppressed intra-molecular

cyclization up to 15% yield. All the TFA-L-/D-Phe-arenes synthesized from TFA-L-/D-Phe-

OSu retained their chirality and the reaction was easier to handle compared to phenylalanine

acid chloride.101

112

Table 26 Friedel–Crafts reaction of TFA-L-/D-Phe-OSu (L-/D-82) into various arenes (40–44) catalyzed by AlCl3

Entry Material

82 Arene TFA-L-Ile-Ar

Inter-molecular

product

TFA-L-/D-Phe-Ar

(%Yield)

Intra-molecular

product

TFA-L-/D-cPhe

(L-/D-84, %Yield)

1 L-

40

TFA-L-/D-Phe-Ph(2-Me) (L-/D-87)

(17:1)

52 21

2 D- 58 26

3 L-

41

TFA-L-/D-Phe-Ph(2-OMe) (L-/D-88)

(13:1)

68 –

4 D- 66 –

113

Entry

(cont’)

Material

82 Arene TFA-L-Ile-Ar

Inter-molecular

product

TFA-L-/D-Phe-Ar

(%Yield)

Intra-molecular

product

TFA-L-/D-cPhe

(L-/D-84, %Yield)

5 L-

42

70 15

6 D- 69 10

7 L-

43

49 36

8 D- 44 33

9 L-

44

31 54

10 D- 36 43

114

To broaden the application of N-hydroxysuccinimide ester, TFA-L-/D-Phe-OSu (L-/D-

82) were utilized for N-heterocycles of N-pyrrole 92 and N-methylpyrrole 93 (Scheme 30).

Previously, N-TFA-L-phenylalanine-benzotriazole was reported as the only N-pyrrole and N-

methylpyrrole acylation products, with no intra-molecular cyclization product.30

Similarly,

when the optical active TFA-L-/D-Phe-OSu (L-/D-82) were treated with N-pyrrole 92, TFA-L-

/D-Phe-(2-Pyr)30

(L-/D-94, Scheme 30 (a)), resulting in a moderate yield in which acylation

occurred specifically at the C-2 position. Regarding acylation into N-methylpyrrole 93, two

isomers of C-2 position-acylated product of TFA-L-/D-Phe-(2-(N-Me)Pyr) (L-/D-95)30

as the

major one and C-3 position-acylated product of TFA-L-/D-Phe(3-(N-Me)Pyr) (L-/D-96) were

found that can be detected by TLC. N-Methyl protecting group of N-methylpyrrole 93 might

trigger further acylation at C-3 position. However, unlike utilization of N-TFA-L-

phenylalanine-benzotriazole30

that needed CH2Cl2 as solvent to undergo the reaction, TFA-L-

/D-Phe-OSu (L-/D-82) showed high solubility towards excess N-pyrrole and N-methylpyrrole.

This indicates the efficiency of TFA-L-/D-Phe-OSu (L-/D-82) as a representative acyl donor

for acylation reaction.

Scheme 30 Friedel–Crafts reaction of TFA-L-/D-Phe-OSu (L-/D-82) into N-pyrrole 92 and N-

methylpyrrole 93 catalyzed by AlCl3

Based on 1H-NMR, the occurrence of overlapping two protons at 3- and 5-positions of

N-methylpyrrole30

was observed for C-2 position at δH 6.62 ppm (d, J = 2.0 Hz) of the

acylated product L-/D-95 (Figure 27). Meanwhile, the proton signal of the 2-position of N-

methylpyrrole for L-/D-96 was shown at δH 6.93 ppm as a singlet and the proton signal of the

5-position of N-methylpyrrole for L-/D-96 appeared at slightly upfield region (δH 6.20, dd, J =

4.3, 2.3 Hz, Figure 27). The 13

C-NMR supports the assignment demonstrated by the

observation of C=O at δC 189.8 ppm signal for the C-2 position-acylated product L-/D-95.30

Meanwhile, the C-3 position-acylated product L-/D-96 showed slightly upperfield carbonyl

115

carbon signal at δC 185.0 ppm. Both utilization of optical active TFA-L-/D-Phe-OSu (L-/D-82)

to 92 and N-methylpyrrole 93 showed no detection of intra-molecular adduct, thus indicating

that these acyl acceptors react relatively fast with the active intermediate to giving TFA-α-

amino-heterocycles ketones.

Figure 27 Selected 1H-NMR (270 MHz, CDCl3) of TFA-L-Phe-(2-(N-Me)Pyr) (L-95) and

TFA-L-Phe(3-(N-Me)Pyr) (L-96)

Synthesis and application of TFA-L-/D-tyrosine-N-hydroxysuccinimide esters

Utilization of the N-hydroxysuccinimide ester (OSu) as a potential acyl donor for aryl-

ketones synthesis was extended to tyrosine derivatives. Following the optical active L-/D-

tyrosine (L-/D-Tyr, L-/D-97) underwent N-TFA protection to result in TFA-L-/D-Tyr (L-/D-98,

Scheme 31), TFA-protection for L-/D-97 utilized a high amount of reagents compare with

other -amino acids (3 equiv. of ethyl trifluoroacetate in the presence of 8 equiv. of

triethylamine in methanol). This can be due to the presence of hydroxy groups in aromatic

moiety of tyrosine which influenced the acidity of the reaction system and hampered TFA

protection in its N-terminal.

Scheme 31 Synthesis of TFA-L-/D-Tyr (L-/D-98)

H-3H-5

H-2 H-5

116

The resultant L-/D-98 were then directly transformed to TFA-L-/D-Tyr-OSu (L-/D-99) by

utilization of NHS and WSCD-HCl in acetone (Table 27). The attempt to increase the

formation of L-/D-99 was conducted as shown in Table 27. When the amount of NHS was

increased and WSCD-HCl amount was kept the same amounts of 1.0 equiv., the desired L-/D-

99 was isolated after purification with 0.1 M NaHCO3 (Table 27 Entries 1–4, 7–8, and 11–12).

Unfortunately, the products were contaminated with unidentifed compounds which were

detected by 1H-NMR. Next, the trial for isolating the pure L-/D-99 was done by increasing the

proportion of NHS (Table 27 Entries 7–10) and found that the contaminant still yielded and it

was difficult to remove. From these results, it was suggested that purification of the products

by 0.1 M NaHCO3 is too harsh condition for L-/D-99 formation. Thus, the purification process

with 0.01 M NaHCO3 was conducted (Table 27 Entries 5–6), and it resulted in L-/D-99 in a

fine yield and readily to be use for Friedel–Crafts acylation.

Table 27 Synthesis of N-TFA-L-/D-tyrosine succinimide ester derivatives (TFA-L-/D-Tyr-

OSu, L-/D-99)

Entrya

Start Material

98

NHS

(equiv.)

WSCD-HCl

(equiv.) Condition

L-/D-99

(%Yield)

1 L-c 1.1 1.0 2h, rt 66%

g

2 D-c 1.1 1.0 2h, rt 70%

g

3 L-d 1.3 1.0 2h, rt 60%

g

4 D-d 1.3 1.0 2h, rt 66%

g

5b L-

d 1.3 1.0 3h, rt 75%

6b D-

d 1.3 1.0 3h, rt 74%

7 L-e 1.3 1.0

f 2h, rt 70%

g

8 D-e 1.3 1.0

f 2h, rt 72%

g

9 L-c 1.3 1.4 2h, rt 68%

g

10 D-c 1.3 1.4 2h, rt 63%

g

11 L-c 1.8 1.0 2h, rt 58%

g

12 D-c 1.8 1.0 2h, rt 54%

g

a Solvent was removed under reduced pressure. Then, residue was dissolve in ethyl acetate and

washed by sat. NaCl and 0.1 M NaHCO3 in H2O. b Solvent was removed under reduced pressure.

Then, residue was dissolve in ethyl acetate and washed by sat. NaCl and 0.01 M NaHCO3 in H2O.

c Conc. 0.08 M in acetone. d Conc. 0.04 M in acetone. e Conc. 0.06 M in acetone. f WSCD-HCl was

dissolved in CH2Cl2 (0.06 M) before addition at 0 oC. g Contaminated with unidentified

compounds, observed by appearance of unidentical α-proton signals in 1H-NMR (ratio

1.00:0.01~0.18).

117

Table 28 shows the Friedel–Crafts reaction of TFA-L-/D-Tyr-OSu (L-/D-99) into benzene

34 catalyzed by AlCl3. The hydroxy group in an aromatic moiety of TFA-L-/D-Tyr-OSu (L-/D-

99) was not hampered in its reactivity to undergo acylation into benzene. Unlike TFA-L-/D-

Phe-OSu (L-/D-82) utilization to benzene (Table 25), the TFA-L-/D-Tyr-Ph (L-/D-100, Table

28) utilization showed no appearance of intra-molecular product. When 6 equiv. AlCl3 was

used, the lower yield of TFA-L-Tyr-Ph (L-100, Table 28, Entry 1) was obtained, due to

formation of a decarbonylated compound 101. Decarbonylated compound 101 can

significantly be determined by 13

C-NMR in which no C=O signals at downfield region were

apparent in comparison with TFA-L-Tyr-Ph (L-100, Figure 28).

A higher proportion of AlCl3 (24 equiv., Table 28, Entry 3–4) suppressed 101 formation

and the resultant TFA-L-/D-Tyr-Ph (L-/D-100) was in a satisfactory yield without loss of

chirality. Decarbonylated compound 101 at low proportion AlCl3 might be due to activation of

carbonyl groups of L-/D-99, faster than cleavage of OSu groups. Moreover, although the

mechanism is still unknown, but it can be confirmed that decarbonylated compound 101 has

lost its optical acivity property. Nevertheless, under an acidic condition offered by the system,

hydroxy substituent in an aromatic moiety of tyrosine skeleton might be unnecessary to be

protected, and utilization of TFA-L-/D-Tyr-OSu (L-/D-99) would be considerably more

effective for direct synthesis of TFA-L-/D-Tyr-Ph (L-/D-100).

Table 28 Friedel–Crafts reaction of TFA-L-/D-Tyr-OSu (L-/D-99) into benzene catalyzed by

AlCl3

Entry Material

99

AlCl3

(equiv.) 100 (%Yield) Ratio of 100 : 101

a

1 L- 6 49% 1.00:0.45

2 L- 12 56% 1.00:0.21

3 L- 24 78% 1.00:0.13

4 D- 24 79% 1.00:0.10 aObserved by

1H-NMR

118

Figure 28 Selected 13

C NMR (67.5 MHz, acetone-d6) comparison of TFA-L-Tyr-Ph (L-100)

with decarbonylated compound 101

3.3 Experimental section

All reagents used were of analytical grade. FT-IR (Fourier-transform infrared

spectroscopy) spectra were recorded on a FT-IR 4100 spectrometer (JASCO, Tokyo, Japan).

NMR spectra were measured by an EX 270 spectrometer (JEOL, Tokyo, Japan). Optical

rotations were measured at 23 °C on a JASCO DIP370 polarimeter (JASCO, Tokyo, Japan).

HRMS-ESI spectra were obtained with a Waters UPLC ESI-TOF mass spectrometer (Waters,

Milford, CT, USA).

3.3.1 General procedure for the preparation of TFA--amino acid

The TFA-α-amino acid was prepared by reported procedure92,93

with slightly

modification. Triethylamine (33 mmol, 1.5 equiv.) was added to a solution of α-amino acid

(22 mmol) in MeOH (22 mL). After 5 min, ethyl trifluoroacetate (29 mmol, 1.3 equiv.) was

added and the reaction mixture was allowed to stir for 24 h. The solvent was removed by

rotary evaporation and the residue remained was dissolved in H2O and acidified with

concentrated HCl. The resulting mixture was extracted with ethyl acetate for several times

and the organic layers combined were washed with brine, dried by MgSO4, filtered, and

C=O

region

119

concentrated by rotary evaporation. Further subjection to high vacuum for overnight, if

needed to solidify the product.

(2S,3S)-3-Methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-L-Ile, L-32):91,103

Colorless amorphous mass. []D = ＋55 (c 1.0, CHCl3). IR (neat) : 3294, 2968, 1740, 1694

cm–1

. 1H-NMR (270 MHz, CDCl3) δ: 10.58 (br s, 1H, COOH), 6.86 (d, J = 8.2 Hz, 1H, NH),

4.68 (dd, J = 8.4, 4.5 Hz, 1H, CHNH), 2.13–1.98 (m, 1H, CHCH3), 1.60–1.44 (m, 1H,

CH2CH3), 1.36–1.19 (m, 1H, CH2CH3), 1.01–0.94 (m, 6H, 2 x CH3) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 175.4, 157.2 (q, 2JCF = 38.0 Hz), 115.6 (q,

1JCF = 287.7 Hz), 56.8, 37.6, 24.9,

15.2, 11.4 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848, found 228.0858.

(2R,3R)-3-Methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-D-Ile, D-32):

Colorless amorphous mass. []D = －55 (c 1.0, CHCl3). IR (neat) : 3293, 2973, 1740, 1699

cm–1

. 1H-NMR (270 MHz, CDCl3) δ: 9.47 (br s, 1H, COOH), 6.79 (d, J = 7.9 Hz, 1H, NH),

4.68 (dd, J = 8.4, 4.5 Hz, 1H, CHNH), 2.12–1.99 (m, 1H, CHCH3), 1.61–1.44 (m, 1H,

CH2CH3), 1.36–1.19 (m, 1H, CH2CH3), 1.01–0.95 (m, 6H, 2 x CH3) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 175.3, 157.3 (q, 2JCF = 37.8 Hz), 115.6 (q,

1JCF = 287.5 Hz), 56.8, 37.6, 24.9,

15.1, 11.3 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848, found 228.0850.

(2S,3R)-3-Methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-L-allo-Ile, L-37):

Colorless amorphous mass. []D = +24 (c 1.0, CHCl3). IR (neat) : 3287, 2971, 1719 cm–1

.

1H-NMR (270 MHz, CDCl3) δ: 8.70 (br s, 1H, CHCOOH), 6.77 (d, J = 8.2 Hz, 1H, NH), 4.76

(dd, J = 8.6, 3.6 Hz, 1H, CHNH), 2.17–2.05 (m, 1H, CHCH3), 1.53–1.38 (m, 1H, CH2CH3),

1.33–1.17 (m, 1H, CH2CH3), 1.01–0.90 (m, 6H, 2 x CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3)

δ: 175.6, 157.4 (q, 2JCF = 37.8 Hz), 115.7 (q,

1JCF = 287.5 Hz), 55.8, 37.6, 26.1, 14.3, 11.5

ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848, found 228.0852.

(2R,3S)-3-Methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-D-allo-Ile, D-37):

Colorless amorphous mass. []D = –24 (c 1.0, CHCl3). IR (neat) : 3302, 2971, 1708 cm–1

.

1H-NMR (270 MHz, CDCl3) δ: 9.12 (br s, 1H, CHCOOH), 6.93 (d, J = 8.6 Hz, 1H, NH), 4.76

(dd, J = 8.9, 3.6 Hz, 1H, CHNH), 2.18–2.04 (m, 1H, CHCH3), 1.53–1.37 (m, 1H, CH2CH3),

1.33–1.17 (m, 1H, CH2CH3), 1.01–0.94 (m, 6H, 2 x CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3)

δ: 175.7, 157.4 (q, 2JCF = 38.0 Hz), 115.7 (q,

1JCF = 287.7 Hz), 55.8, 37.5, 26.1, 14.3, 11.5

ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848, found 228.0851.

2-(2,2,2-Trifluoroacetamido)acetic acid (TFA-Gly, 52):103–105

Colorless amorphous mass.

IR (neat) : 3299, 2992, 1682 cm–1

. 1H-NMR (270 MHz, CD3OD) δ: 4.01 (s, 2H, CH2NH)

ppm. 13

C NMR (67.5 MHz, CD3OD) δ: 171.5, 159.4 (q, 2JCF = 37.4 Hz), 117.4 (q,

1JCF =

120

286.2 Hz), 41.7 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C4H5F3NO3 172.0222, found

172.0241.

(S)-2-(2,2,2-Trifluoroacetamido)propanoic acid (TFA-L-Ala, L-56):103,106,107

Colorless

amorphous mass. []D = +38 (c 1.0, CHCl3). IR (neat) : 3330, 2952, 1752 cm–1

. 1H-NMR

(270 MHz, CDCl3) δ: 9.23 (br s, 1H, COOH), 7.00 (br s, 1H, NH), 4.72–4.62 (m, 1H,

CHCH3), 1.58 (d, J = 7.3 Hz, 3H, CHCH3) ppm. 13

C NMR (67.5 MHz, CDCl3) δ: 176.3,

156.9 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.9 Hz), 48.5, 17.6 ppm. HRMS-ESI (m/z) [M +

H]+ calcd for C5H7F3NO3 186.0378, found 186.0389.

(R)-2-(2,2,2-Trifluoroacetamido)propanoic acid (TFA-D-Ala, D-56):107

Colorless

amorphous mass. []D = –38 (c 1.0, CHCl3). IR (neat) : 3295, 2949, 1756 cm–1

. 1H-NMR

(270 MHz, CDCl3) δ: 8.96 (br s, 1H, COOH), 6.99 (br s, 1H, NH), 4.73–4.62 (m, 1H,

CHCH3), 1.58 (d, J = 7.3 Hz, 3H, CHCH3) ppm. 13

C NMR (67.5 MHz, CDCl3) δ: 176.0,

157.1 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.2 Hz), 48.5, 17.2 ppm. HRMS-ESI (m/z) [M +

H]+ calcd for C5H7F3NO3 186.0378, found 186.0365.

(S)-3-Methyl-2-(2,2,2-trifluoroacetamido)butanoic acid (TFA-L-Val, L-60): 103,104,106

Colorless amorphous mass. []D = +53 (c 1.0, CHCl3). IR (neat) : 3286, 2969, 1739 cm–1

.

1H-NMR (270 MHz, CD3Cl3) δ: 10.35 (br s, 1H, CHCOOH), 6.81 (d, J = 7.9 Hz, 1H, NH),

4.65 (dd, J = 8.6, 4.6 Hz, 1H, CHNH), 2.41–2.29 (m, 1H, CHCH3), 1.05–0.99 (m, 5H, 2 x

CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 175.5, 157.3 (q, 2JCF = 36.3 Hz), 115.7 (q,

1JCF =

287.2 Hz), 57.4, 31.1, 18.7, 17.4 ppm. HRMS-ESI (m/z) [M + Na]+ calcd for C7H10F3NO3Na

236.0510, found 236.0520.

(R)-3-Methyl-2-(2,2,2-trifluoroacetamido)butanoic acid (TFA-D-Val, D-60):104

Colorless

amorphous mass. []D = –53 (c 1.0, CHCl3). IR (neat) : 3295, 2970, 1753 cm–1

. 1H-NMR

(270 MHz, CD3Cl3) δ: 10.99 (br s, 1H, CHCOOH), 6.89 (d, J = 8.6 Hz, 1H, NH), 4.64 (dd, J

= 8.6, 4.6 Hz, 1H, CHNH), 2.41–2.26 (m, 1H, CHCH3), 1.05–0.99 (m, 6H, 2 x CH3) ppm.

13C-NMR (67.5 MHz, CDCl3) δ: 175.5, 157.3 (q,

2JCF = 37.4 Hz), 115.6 (q,

1JCF = 287.7 Hz),

57.4, 31.1, 18.7, 17.4 ppm. HRMS-ESI (m/z) [M + Na]+ calcd for C7H10F3NO3Na 236.0510,

found 236.0518

(S)-4-methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-L-Leu, L-64):91,104,108

Colorless amorphous mass. []D = +24 (c 1.0, CHCl3). IR (neat) : 3294, 2963, 1731 cm–1

.

1H-NMR (270 MHz, CD3Cl3) δ: 8.90 (br s, 1H, CHCOOH), 6.78 (br s, 1H, NH), 4.74–4.65

(m, 1H, CHNH), 1.88–1.64 (m, 3H, CH2CH), 1.00 (s, 3H, CH3), 0.98 (s, 3H, CH3) ppm. 13

C-

NMR (67.5 MHz, CDCl3) δ: 176.4, 157.2 (q, 2JCF = 38.0 Hz), 115.6 (q,

1JCF = 287.2 Hz), 51.1,

121

40.8, 24.8, 22.6, 21.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848,

found 228.0859.

(R)-4-Methyl-2-(2,2,2-trifluoroacetamido)pentanoic acid (TFA-D-Leu, D-64):104

Colorless

amorphous mass. []D = –24 (c 1.0, CHCl3). IR (neat) : 3300, 2965, 1733 cm–1

. 1H-NMR

(270 MHz, CD3Cl3) δ: 8.54 (br s, 1H, CHCOOH), 6.73 (d, J = 7.6 Hz, 1H, NH), 4.74–4.65 (m,

1H, CHNH), 1.88–1.61 (m, 3H, CH2CH), 1.00 (s, 3H, CH3), 0.98 (s, 3H, CH3) ppm. 13

C-

NMR (67.5 MHz, CDCl3) δ: 175.9, 157.8 (q, 2JCF = 38.0 Hz), 115.6 (q,

1JCF = 287.0 Hz), 51.2,

40.2, 24.7, 22.4, 21.2 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H13F3NO3 228.0848,

found 228.0865.

(S)-2-(2,2,2-Trifluoroacetamido)pentanoic acid (TFA-L-Nva, L-68):104

Colorless

amorphous mass. []D = +58 (c 1.0, CHCl3). IR (neat) : 3292 cm–1

, 2967, 1732, 1696 cm–1

.

1H-NMR (270 MHz, CDCl3) δ: 6.76 (d, J = 6.9 Hz, 1H, NH), 4.69 (td, J = 7.3, 5.4 Hz, 1H,

CHNH), 2.06–1.92 (m, 1H, CHCH2), 1.88–1.74 (m, 1H, CHCH2), 1.50–1.35 (m, 2H,

CH2CH3), 0.98 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 176.0,

157.3 (q, 2JCF = 37.8 Hz), 115.6 (q,

1JCF = 287.3 Hz), 52.4, 33.5, 18.4, 13.3 ppm. HRMS-ESI

(m/z) [M + H]+ calcd for C7H11F3NO3 214.0691, found 214.0693.

(R)-2-(2,2,2-Trifluoroacetamido)pentanoic acid (TFA-D-Nva, D-68):104

Colorless

amorphous mass. []D = –58 (c 1.0, CHCl3). IR (neat) : 3319, 2969, 1745, 1695 cm–1

. 1H-

NMR (270 MHz, CDCl3) δ: 6.75 (d, J = 6.6 Hz, 1H, NH), 4.69 (td, J = 7.5, 5.2 Hz, 1H,

CHNH), 2.06–1.92 (m, 1H, CHCH2), 1.88–1.74 (m, 1H, CHCH2), 1.57–1.32 (m, 2H,

CH2CH3), 0.98 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 176.2,

157.2 (q, 2JCF = 38.0 Hz), 115.6 (q,

1JCF = 287.3 Hz), 52.4, 33.6, 18.4, 13.4 ppm. HRMS-ESI

(m/z) [M + H]+ calcd for C7H11F3NO3 214.0691, found 214.0696.

(S)-2-(2,2,2-Trifluoroacetamido)hexanoic acid (TFA-L-Nle, L-72):104

Colorless amorphous

mass. []D = +67 (c 1.0, CHCl3). IR (neat) : 3314, 2936, 1728 cm–1

. 1H-NMR (270 MHz,

CDCl3) δ: 6.73 (br s, 1H, NH), 4.67 (td, J = 7.4, 5.4 Hz, 1H, CHNH), 2.08–1.94 (m, 1H,

CHCH2), 1.89–1.75 (m, 1H, CHCH2), 1.42–1.26 (m, 4H, 2 x CH2), 0.92 (t, J = 6.9 Hz, 3H,

CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 176.1, 157.2 (q, 2JCF = 38.0 Hz), 115.6 (q,

1JCF = 287.5 Hz), 52.6, 31.3, 27.0, 22.1, 13.6 ppm. HRMS-ESI (m/z) [M + H]

+ calcd for

C8H13F3NO3 228.0848, found 228.0850.

(R)-2-(2,2,2-Trifluoroacetamido)hexanoic acid (TFA-D-Nle, D-72):104

Colorless

amorphous mass. []D = –67 (c 1.0, CHCl3). IR (neat) : 3302, 2936, 1740 cm–1

. 1H-NMR

(270 MHz, CDCl3) δ: 6.74 (br s, 1H, NH), 4.69 (td, J = 7.4, 5.4 Hz, 1H, CHNH), 2.06–1.94

(m, 1H, CHCH2), 1.89–1.75 (m, 1H, CHCH2), 1.41–1.31 (m, 4H, 2 x CH2), 0.92 (t, J = 6.9 Hz,

122

3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 176.2, 157.1 (q, 2JCF = 38.0 Hz), 115.6

(q, 1JCF = 287.7 Hz), 52.5, 31.4, 27.0, 22.1, 13.6 ppm. HRMS-ESI (m/z) [M + H]

+ calcd for

C8H13F3NO3 228.0848, found 228.0843.

(S)-1-(2,2,2-Trifluoroacetyl)pyrrolidine-2-carboxylic acid (TFA-L-Pro, L-76): Colorless

amorphous mass. [α]D = –86 (c 1.0, CHCl3); Lit. 99

[α]D = –65.19 (c 1.08, benzene). IR (neat)

: 1739, 1705 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 4.59 (1H, dd, J = 8.4, 3.8 Hz, CH2CH),

3.90–3.69 (2H, m, CH2), 2.36–1.99 (4H, m, 2 x CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ:

[176.2 & 176.0], 156.1 (q, 2JCF = 37.8 Hz), 116.0 (q,

1JCF = 287.0 Hz), [60.0 & 59.1], 48.0 &

47.2], [31.5 & 28.3], [24.8 & 21.0] ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd

for C7H9F3NO3 212.0535, found 212.0536.

(R)-1-(2,2,2-Trifluoroacetyl)pyrrolidine-2-carboxylic acid (TFA-D-Pro, D-76): Colorless

amorphous mass. [α]D = +86 (c 1.0, CHCl3). IR (neat) : 1738, 1709 cm-1

. 1H-NMR (270

MHz, CDCl3) δ: 4.59 (1H, dd, J = 8.2, 3.6 Hz, CH2CH), 3.87–3.67 (2H, m, CH2), 2.37–1.96

(4H, m, 2 x CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: [175.9 & 175.7], 156.1 (q, 2JCF =

37.8 Hz), 116.0 (q, 1JCF = 286.8 Hz), [60.0 & 59.1], [48.0 & 47.2], [31.5 & 28.3], [24.8 &

21.0] ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd for C7H9F3NO3 212.0535,

found 212.0555.

(S)-3-phenyl-2-(2,2,2-trifluoroacetamido)propanoic acid (TFA-L-Phe, L-81): Colorless

amorphous mass. [α]D = +13 (c 1.0, MeOH); Lit. 103

[α]D = +17.2 (c 2, ethanol). IR (neat) :

3319, 3099, 3033, 2929, 1739, 1706 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.38–7.30 (3H, m,

Ar-H), 7.14 (2H, d, J = 7.6 Hz, Ar-H), 6.70 (1H, br d, J = 5.6 Hz, NH), 4.94 (1H, q, J = 5.6,

5.6, 5.6 Hz, CHNH), 3.31 (1H, dd, J = 14.2, 5.6 Hz, CH2CH), 3.22 (1H, dd, J = 14.2, 5.6 Hz,

CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 174.2, 156.7 (q, 2JCF = 38.2 Hz), 134.2, 129.2

(2 x CH), 129.0 (2 x CH), 127.8, 115.5 (q, 1JCF = 287.2 Hz), 53.2, 36.9 ppm. HRMS-ESI

(m/z) [M+H]+ calcd for C11H11F3NO3 262.0691, found 262.0699.

(R)-3-phenyl-2-(2,2,2-trifluoroacetamido)propanoic acid (TFA-D-Phe, D-81): Colorless

amorphous mass. [α]D = –13 (c 1.0, MeOH); Lit.104

[α]D = –17.2 (c 2, ethanol). IR (neat) :

3320, 3093, 3033, 2934, 1740, 1702 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.34–7.30 (3H, m,

Ar-H), 7.14 (2H, d, J = 7.3 Hz, Ar-H), 6.68 (1H, br d, J = 5.6 Hz, NH), 4.95 (1H, q, J = 5.6,

5.6, 5.6 Hz, CHNH), 3.32 (1H, dd, J = 14.2, 5.6 Hz, CH2CH), 3.22 (1H, dd, J = 14.2, 5.6 Hz,

CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 174.6, 156.8 (q, 2JCF = 38.4 Hz), 134.2, 129.2

(2 x CH), 129.0 (2 x CH), 127.8, 115.5 (q, 1JCF = 287.3 Hz), 53.3, 36.9 ppm. HRMS-ESI

(m/z) [M+H]+ calcd for C11H11F3NO3 262.0691, found 262.0694.

123

3.3.2 Procedure for the preparation of TFA-L-/D-tyrosine (L-/D-98)

Triethylamine (136 mmol, 8 equiv.) was added to a solution of α-amino acid (17 mmol)

in MeOH (40 mL). After 5 min, ethyl trifluoroacetate (51 mmol, 3 equiv.) was added and the

reaction was allowed to stir for 7 d. The solvent was removed by rotary evaporation and the

residue that remained was dissolved in H2O and acidified with concentrated HCl.

The mixture was extracted with ethyl acetate for several times and the organic layers

were combined and washed with brine, dried by MgSO4, filtered, and concentrated by rotary

evaporation. Further subjection into high vacuum for overnight, if needed to solidify the

product (TFA-L-/D-tyrosine, L-/D-98).

(S)-3-(4-hydroxyphenyl)-2-(2,2,2-trifluoroacetamido)propanoic acid (TFA-L-Tyr, L-98):

Yellowish amorphous mass. [α]D = +26 (c 1.0, MeOH). IR (neat) : 3302, 3098, 2932, 1734

cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.55 (1H, br s, OH), 7.19 (2H, d, J = 8.2 Hz,

Ar-H), 6.83 (2H, d, J = 8.6 Hz, Ar-H), 4.78 (1H, td, J = 9.7, 4.6 Hz, CHNH), 3.31 (1H, dd, J

= 14.2, 4.6 Hz, CH2CH), 3.08 (1H, dd, J = 14.2, 9.7 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz,

ACETONE-D6) δ: 171.6, 157.3 (q, 2JCF = 36.9 Hz), 157.1, 131.0 (2 x CH), 128.2, 116.8 (q,

1JCF = 287.2 Hz), 116.0 (2 x CH), 55.0, 36.4 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd for

C11H10F3NO4Na 300.0460, found 300.0444.

(R)-3-(4-hydroxyphenyl)-2-(2,2,2-trifluoroacetamido)propanoic acid (TFA-D-Tyr, D-98):

Yellowish amorphous mass. [α]D = –26 (c 1.0, MeOH). IR (neat) : 3295, 3098, 2932, 1732

cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.51 (1H, br s, OH), 7.15 (2H, d, J = 8.2 Hz,

Ar-H), 6.80 (2H, d, J = 8.2 Hz, Ar-H), 4.78 (1H, td, J = 9.6, 4.9 Hz, CHNH), 3.29 (1H, dd, J

= 14.2, 4.9 Hz, CH2CH), 3.05 (1H, dd, J = 14.2, 9.6 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz,

ACETONE-D6) δ: 171.9, 157.5 (q, 2JCF = 37.1 Hz), 157.0, 131.0 (2 x CH), 128.1, 116.8 (q,

1JCF = 287.3 Hz), 116.1 0 (2 x CH), 55.0, 36.4 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd for

C11H10F3NO4Na 300.0460, found 300.0469.

3.3.3 General Procedure for the Preparation of TFA--Amino Acid N-Hydroxysuccinimide

Ester

NHS (N-hydroxysuccinimide, 1.1 equiv.) was added to a solution of TFA--amino acid

(1.0 mmol) in pre-cooled CH2Cl2 (10 mL). The suspension of WSCD-HCl (water soluble

carbodiimide hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

monohydrochloride, 1.0 equiv.) in CH2Cl2 (10 mL) was added drop-wise at 0 °C and the

reaction was stirred for 2–4 h. The solvent was removed by rotary evaporation. The residue

that remained was dissolved in ethyl acetate; washed with water, sat. NaHCO3, sat. NaCl,

124

dried over MgSO4, and then evaporated. The product was solidified by washing with hexane

to be used for further reaction. For TFA--Amino Acid N-Hydroxysuccinimide Ester 53

detail experiment are refer to Scheme 16; L-/D-57 detail experiment are refer to Table 24; L-

/D-69 detail experiment are refer to Scheme 23; L-/D-73 detail experiment are refer to Scheme

26; L-/D-99 detail experiment are refer to Table 27.

(2S,3S)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)pentanoate (TFA-

L-Ile-OSu, L-33):109

Colorless amorphous mass. [α]D = –4.0 (c 1.0, CHCl3); ref.109

[α]D =

−63.6 (c 1, CH3OH). IR (neat) ν: 3265, 2965, 1785, 1740 cm

−1.

1H-NMR (270 MHz, CDCl3)

δ: 7.41 (d, J = 8.6 Hz, 1H, NH), 4.97 (dd, J = 8.6, 5.3 Hz, 1H, CHNH), 2.89 (s, 4H, 2 x CH2),

2.23–2.08 (m, 1H, CHCH3), 1.75–1.60 (1H, m, CH2CH3), 1.45–1.29 (1H, m, CH2CH3), 1.11

(d, J = 6.9 Hz, 3H, CHCH3), 1.02 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz,

CDCl3) δ: 168.5 (2 x CO), 166.3, 156.9 (q, 2JCF = 38.4 Hz), 115.5 (q,

1JCF = 287.9 Hz), 55.3,

38.0, 25.5 (2 x CH2), 24.7, 14.8, 11.3 ppm. HRMS-ESI (m/z) [M + Na]+ calcd for

C12H15F3N2O5Na 347.0831, found 347.0822.

(2R,3R)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)pentanoate

(TFA-D-Ile-OSu, D-33): Colorless amorphous mass. [α]D = +4.0 (c 1.0, CHCl3). IR (neat) ν:

3285, 2968, 1789, 1731 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 6.97 (d, J = 8.2 Hz, 1H, NH),

4.97 (dd, J = 8.6, 4.9 Hz, 1H, CHNH), 2.86 (s, 4H, 2 x CH2) 2.20–2.05 (m, 1H, CHCH3),

1.71–1.56 (m, 1H, CH2CH3), 1.41–1.21 (m, 1H, CH2CH3), 1.07 (d, J = 6.9 Hz, 3H, CHCH3),

1.00 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C NMR (67.5 MHz, CDCl3) δ: 168.7 (2 x CO),

166.2, 156.9 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.5 Hz), 55.3, 37.7, 25.4 (2 x CH2), 24.6,

14.7, 11.1 ppm. HRMS-ESI (m/z) [M + Na]+ calcd for C12H15F3N2O5Na 347.0831, found

347.0833.

(2S,3R)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)pentanoate (TFA-

L-allo-Ile-OSu, L-38): Colorless amorphous mass. [α]D = –4.0 (c 1.0, CHCl3). IR (neat) ν:

3316, 2929, 1788, 1752 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.06 (d, J = 9.2 Hz, 1H, NH),

5.10 (dd, J = 9.2, 4.0 Hz, 1H, CHNH), 2.86 (s, 4H, 2 x CH2), 2.31–2.15 (m, 1H, CHCH3),

1.54–1.41 (m, 1H, CH2CH3), 1.37–1.23 (m, 1H, CH2CH3), 1.05 (d, J = 6.9 Hz, 3H, CHCH3),

1.00 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.4 (2 x CO),

166.8, 157.0 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.7 Hz), 54.1, 38.2, 25.9, 25.5 (2 x CH2),

14.1, 11.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C12H16F3N2O5 325.1011, found

325.1013.

125

(2R,3S)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)pentanoate (TFA-

D-allo-Ile-OSu, D-38): Colorless amorphous mass. [α]D = +4.0 (c 1.0, CHCl3). IR (neat) ν:

3327, 2971, 1752 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.07 (d, J = 9.2 Hz, 1H, NH), 5.10

(dd, J = 9.1, 4.1 Hz, 1H, CHNH), 2.86 (s, 4H, 2 x CH2), 2.28–2.17 (m, 1H, CHCH3), 1.57–

1.39 (m, 1H, CH2CH3), 1.37–1.22 (m, 1H, CH2CH3), 1.05 (d, J = 6.9 Hz, 3H, CHCH3), 1.00 (t,

J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.7 (2 x CO), 166.7, 157.1

(q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.3 Hz), 54.1, 37.9, 25.8, 25.4 (2 x CH2), 14.0, 11.4

ppm. HRMS-ESI (m/z) [M + H]+ calcd for C12H16F3N2O5 325.1011, found 325.1014.

2,5-Dioxocyclopentyl 2-(2,2,2-trifluoroacetamido)acetate (TFA-Gly-OSu, 53) 105

:

Colorless amorphous mass. IR (neat) ν: 3313, 2998, 1690 cm−1

. 1H-NMR (270 MHz, CD3OD)

δ: 4.44 (s, 2H, CH2NH), 2.84 (s, 4H, 2 x CH2) ppm. 13

C NMR (67.5 MHz, ACETONE-D6) δ:

170.1 (2 x CO), 165.7 158.2 (q, 2JCF = 37.4 Hz), 116.8 (q,

1JCF = 287.0 Hz), 39.5, 26.2 (2 x

CH2) ppm. HRMS-ESI (m/z) [M + H]+ calcd for C8H7F3N2O5Na 291.0205, found 291.0208.

(S)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)propanoate (TFA-L-Ala-OSu,

L-57): Colorless amorphous mass. [α]D = −46 (c 1.0, CHCl3). IR (neat) ν: 3332, 2999, 1793,

1734 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.39 (d, J = 7.3 Hz, 1H, NH), 5.08–4.97 (m, 1H,

CHCH3), 2.86 (s, 4H, 2 x CH2), 1.68 (d, J = 7.3 Hz, 3H, CHCH3) ppm. 13

C NMR (67.5 MHz,

CDCl3) δ: 168.8 (2 x CO), 167.4, 156.8 (q, 2JCF = 38.4 Hz), 115.6 (q,

1JCF = 287.2 Hz), 46.7,

25.5 (2 x CH2), 17.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C9H10F3N2O5 283.0542,

found 283.0555.

(R)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)propanoate (TFA-D-Ala-OSu,

D-57): Colorless amorphous mass. [α]D = +46 (c 1.0, CHCl3). IR (neat) ν: 3350, 2999, 1798,

1726 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.73 (d, J = 7.6 Hz, 1H, NH), 5.05–4.94 (m, 1H,

CHCH3), 2.85 (s, 4H, 2 x CH2), 1.66 (d, J = 7.3 Hz, 3H, CHCH3) ppm.13

C NMR (67.5 MHz,

CDCl3) δ: 169.0 (2 x CO), 167.1, 156.8 (q, 2JCF = 38.2 Hz), 115.4 (q,

1JCF = 287.3 Hz), 46.7,

25.4 (2 x CH2), 17.1 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C9H10F3N2O5 283.0542,

found 283.0553.

(S)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)butanoate (TFA-L-

Val-OSu, L-61):109

Colorless amorphous mass. [α]D = −22 (c 1.0, CHCl3); ref. 109

[α]D =

−73.3 (c 1, CH3OH). IR (neat) ν: 3310, 2979, 1796, 1725 cm−1

. 1H-NMR (270 MHz, CD3Cl3)

δ: 7.49 (d, J = 8.9 Hz, 1H, NH), 4.88 (dd, J = 8.7, 5.4 Hz, 1H, CHNH), 2.84 (s, 4H, 2 x CH2),

2.47–2.34 (m, 1H, CHCH3), 1.08 (d, J = 6.9 Hz, 6H, 2 x CH3) ppm. 13

C-NMR (67.5 MHz,

CDCl3) δ: 168.9 (2 x CO), 166.1, 157.1 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.5 H), 55.9,

31.2, 25.4 (2 x CH2), 18.3, 17.2 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C11H14F3N2O5

311.0855, found 311.0858.

126

(R)-2,5-Dioxopyrrolidin-1-yl 3-methyl-2-(2,2,2-trifluoroacetamido)butanoate (TFA-D-

Val-OSu, D-61): Colorless amorphous mass. [α]D = +22 (c 1.0, CHCl3). IR (neat) ν: 3298,

2975, 1725 cm−1

. 1H-NMR (270 MHz, CD3Cl3) δ: 6.93 (d, J = 8.6 Hz, 1H, NH), 4.95 (dd, J =

8.9, 4.9 Hz, 1H, CHNH), 2.87 (s, 4H, 2 x CH2), 2.53–2.35 (m, 1H, CHCH3), 1.09 (d, J = 6.9

Hz, 6H, 2 x CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.4 (2 x CO), 166.4, 157.0 (q, 2JCF

= 38.0 Hz), 115.5 (q, 1JCF = 287.7 Hz), 55.9, 31.7, 25.5 (2 x CH2), 18.4, 17.2 ppm. HRMS-

ESI (m/z) [M + H]+ calcd for C11H14F3N2O5 311.0855, found 311.0857.

(S)-2,5-Dioxopyrrolidin-1-yl 4-methyl-2-(2,2,2-trifluoroacetamido)pentanoate (TFA-L-

Leu-OSu, L-65) 109

: Colorless amorphous mass. [α]D = −42 (c 1.0, CHCl3); ref. 109

[α]D =

−50.8 (c 1, CH3OH). IR (neat) ν: 3295, 2967, 1734, 1712 cm−1

. 1H-NMR (270 MHz, CD3Cl3)

δ: 6.86 (d, J = 8.6 Hz, 1H, NH), 5.04 (td, J = 8.9, 4.9 Hz, 1H, CHNH), 2.86 (s, 4H 2 x CH2),

2.00–1.72 (m, 3H, CH2CH), 1.02 (d, J = 2.3 Hz, 3H, CH3), 1.00 (d, J = 2.3 Hz, 3H, CH3) ppm.

13C-NMR (67.5 MHz, CDCl3) δ 168.6 (2 x CO), 167.2, 156.9 (q,

2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.7 Hz), 49.3, 41.0, 25.5 (2 x CH2), 24.7, 22.6, 21.5 ppm. HRMS-ESI (m/z) [M +

Na]+ calcd for C12H15F3N2O5Na 347.0831, found 347.0832.

(R)-2,5-Dioxopyrrolidin-1-yl 4-methyl-2-(2,2,2-trifluoroacetamido)pentanoate (TFA-D-

Leu-OSu, D-65): Colorless amorphous mass. [α]D = +42 (c 1.0, CHCl3). IR (neat) ν: 3305,

2965, 1733, 1719 cm−1

. 1H-NMR (270 MHz, CD3Cl3) δ: 6.97 (br s, 1H, NH), 5.05 (td, J = 8.7,

4.9 Hz, 1H), 2.86 (s, 4H, 2 x CH2), 2.00–1.74 (m, 3H, CH2CH), 1.03–0.98 (m, 6H, 2 x CH3)

ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.6 (2 x CO), 167.2, 156.9 (q, 2JCF = 38.2 Hz), 115.5

(q, 1JCF = 287.7 Hz), 49.3, 41.0, 25.5 (2 x CH2), 24.7, 22.6, 21.5 ppm. HRMS-ESI (m/z) [M +

Na]+ calcd for C12H15F3N2O5Na 347.0831, found 347.0830.

(S)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)pentanoate (TFA-L-Nva-OSu,

L-69): Colorless amorphous mass. [α]D = −27 (c 1.0, CHCl3). IR (neat) ν: 3325, 2962, 1744,

1711 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 6.83 (d, J = 7.3 Hz, 1H, NH), 5.03 (td, J = 8.0, 5.5

Hz, 1H, CHNH), 2.87 (s, 4H, 2 x CH2), 2.17–2.02 (m, 1H, CHCH2), 1.99–1.85 (m, 1H,

CHCH2), 1.59–1.40 (m, 2H, CH2CH3), 1.01 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR

(67.5 MHz, CDCl3) δ: 168.6 (2 x CO), 166.9, 156.9 (q, 2JCF = 38.0 Hz), 115.5 (q,

1JCF = 287.7

Hz), 50.7, 34.0, 25.5 (2 x CH2), 18.2, 13.3 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C11H14F3N2O5 311.0855, found 311.0856.

(R)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)pentanoate (TFA-D-Nva-OSu,

D-69): Colorless amorphous mass. [α]D = +27 (c 1.0, CHCl3). IR (neat) ν: 3322, 2962, 1744,

1711 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.11 (d, J = 8.2 Hz, 1H, NH), 5.02 (td, J = 8.1, 5.4

Hz, 1H, CHNH), 2.86 (s, 4H, 2 x CH2), 2.15–2.01 (m, 1H, CHCH2), 1.98–1.84 (m, 1H,

CHCH2), 1.60–1.43 (m, 2H, CH2CH3), 1.00 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR

127

(67.5 MHz, CDCl3) δ: 168.6 (2 x CO), 166.9, 156.9 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.9

Hz), 50.7, 34.0, 25.5 (2 x CH2), 18.2, 13.3 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C11H14F3N2O5 311.0855, found 311.0861.

(S)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)hexanoate (TFA-L-Nle-OSu, L-

73): Colorless amorphous mass. [α]D = −18 (c 1.0, CHCl3). IR (neat) ν: 3332, 2958, 1721,

1703 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.34 (d, J = 8.2 Hz, 1H, NH), 4.98 (td, J = 8.2, 5.3

Hz, 1H, CHNH), 2.85 (s, 4H, 2 x CH2), 2.16–2.03 (m, 1H, CHCH2), 1.99–1.85 (m, 1H,

CHCH2), 1.52–1.32 (m, 4H, 2 x CH2), 0.93 (t, J = 7.1 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 168.8 (2 x CO), 166.8, 156.9 (q, 2JCF = 38.2 Hz), 115.5 (q,

1JCF = 287.7 Hz),

50.8, 31.5, 26.9, 25.4 (2 x CH2), 21.9, 13.5 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C12H16F3N2O5 325.1011, found 325.1021.

(R)-2,5-Dioxopyrrolidin-1-yl 2-(2,2,2-trifluoroacetamido)hexanoate (TFA-D-Nle-OSu, D-

73): Colorless amorphous mass. [α]D = +18 (c 1.0, CHCl3). IR (neat) ν: 3324, 2958, 1724,

1708 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 6.89 (br s, 1H, NH), 5.01 (td, J = 7.8, 5.5 Hz, 1H,

CHNH), 2.87 (s, 4H, 2 x CH2), 2.18–2.04 (m, 1H, CHCH2), 2.00–1.86 (m, 1H, CHCH2),

1.52–1.36 (m, 4H, 2 x CH2), 0.94 (t, J = 7.1 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz,

CDCl3) δ: 168.6 (2 x CO), 166.9, 156.9 (q, 2JCF = 38.4 Hz), 115.5 (q,

1JCF = 287.3 Hz), 50.8,

31.7, 26.8, 25.5 (2 x CH2), 21.9, 13.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C12H16F3N2O5 325.1011, found 325.1016.

(S)-2,5-dioxopyrrolidin-1-yl 1-(2,2,2-trifluoroacetyl)pyrrolidine-2-carboxylate (TFA-L-

Pro-OSu, L-77): Colorless amorphous mass. [α]D = –85 (c 1.0, CHCl3); Lit. 109

[α]D = –98 (c

1.0, CH3OH). IR (neat) : 3376, 2988, 1830, 1786, 1758 cm-1

. 1H-NMR (270 MHz, CDCl3)

δ: 4.89 (1H, t, J = 6.3 Hz, CH2CH), 3.95–3.70 (2H, m, CH2), 2.84 (4H, s, 2 x CH2), 2.45–2.35

(2H, m, CH2), 2.30–2.04 (2H, m, CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.6, 166.9,

166.4, 155.9 (q, 2JCF = 38.2 Hz), 115.9 (q,

1JCF = 286.8 Hz), [57.9 & 57.4], [48.0 & 47.1],

[32.1 & 28.7], 25.5, [24.8 & 21.0] ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd

for C11H12F3N2O5 309.0698, found 309.0708.

(R)-2,5-dioxopyrrolidin-1-yl 1-(2,2,2-trifluoroacetyl)pyrrolidine-2-carboxylate (TFA-D-

Pro-OSu, D-77): Colorless amorphous mass. [α]D = +85 (c 1.0, CHCl3). IR (neat) : 3376,

2987, 1827, 1785, 1758 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 4.88 (1H, t, J = 6.1 Hz, CH2CH),

3.92–3.68 (2H, m, CH2), 2.84 (4H, s, 2 x CH2), 2.47–2.35 (2H, m, CH2), 2.26–2.11 (2H, m,

CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.5, 166.8, 166.4, 155.9 (q, 2JCF = 38.2 Hz),

115.9 (q, 1JCF = 287.0 Hz), [58.0 & 57.4], [48.0 & 47.1], [32.1 & 28.7], 25.5, [24.8 & 21.0]

ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd for C11H12F3N2O5 309.0698, found

309.0725.

128

(S)-2,5-dioxopyrrolidin-1-yl 3-phenyl-2-(2,2,2-trifluoroacetamido)propanoate (TFA-L-

Phe-OSu, L-82): Colorless amorphous mass. [α]D = –34 (c 1.0, MeOH). IR (neat) : 3310,

3109, 3033, 2947, 1826, 1818, 1785, 1762, 1737, 1723 cm-1

. 1H-NMR (270 MHz, CDCl3) δ:

7.34–7.27 (5H, m, Ar-H), 6.87 (1H, br d, J = 5.9 Hz, NH), 5.27 (1H, q, J = 5.9, 5.9, 5.9 Hz,

CHNH), 3.42 (1H, dd, J = 14.3, 5.9 Hz, CH2CH), 3.28 (1H, dd, J = 14.3, 5.9 Hz, CH2CH),

2.85 (4H, s, 2 x CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.5 (2 x CO), 166.1, 156.6 (q,

2JCF = 38.0 Hz), 133.4, 129.5 (2 x CH), 128.9 (2 x CH), 127.9, 115.3 (q,

1JCF = 287.2 Hz),

51.5, 37.2, 25.5 (2 x CH2) ppm. HRMS-ESI (m/z) [M+H]+ calcd for C15H14F3N2O5 359.0855,

found 359.0857.

(R)-2,5-dioxopyrrolidin-1-yl 3-phenyl-2-(2,2,2-trifluoroacetamido)propanoate (TFA-D-

Phe-OSu, D-82): Colorless amorphous mass. [α]D = +34 (c 1.0, MeOH). IR (neat) : 3308,

3111, 3033, 2946, 1826, 1816, 1785, 1761, 1735, 1721 cm-1

. 1H-NMR (270 MHz, CDCl3) δ:

7.35–7.28 (5H, m, Ar-H), 6.79 (1H, br d, J = 5.9 NH), 5.28 (1H, q, J = 5.9, 5.9, 5.9 Hz,

CHNH), 3.42 (1H, dd, J = 14.3, 5.9 Hz, CH2CH), 3.30 (1H, dd, J = 14.3, 5.9 Hz, CH2CH),

2.86 (4H, s, 2x CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 168.5 (2 x CO), 166.1, 156.6 (q,

2JCF = 38.4 Hz), 133.3, 129.5 (2 x CH), 128.9 (2 x CH), 127.9, 115.3 (q,

1JCF = 288.1 Hz),

51.5, 37.2, 25.5 (2 x CH2) ppm. HRMS-ESI (m/z) [M+H]+ calcd for C15H14F3N2O5 359.0855,

found 359.0856.

(S)-2,5-dioxopyrrolidin-1-yl 3-(4-hydroxyphenyl)-2-(2,2,2-

trifluoroacetamido)propanoate (TFA-L-Tyr-OSu, L-99): Yellowish amorphous mass. [α]D

= –29 (c 1.0, MeOH). IR (neat) : 3324, 1820, 1792, 1755 cm-1

. 1H-NMR (270 MHz,

ACETONE-D6) δ: 8.86 (1H, d, J = 8.6 Hz, NH), 8.15 (1H, br s, OH), 7.10 (2H, d, J = 8.2 Hz,

Ar-H), 6.67 (2H, d, J = 8.2 Hz, Ar-H), 5.03 (1H, td, J = 10.2, 4.6 Hz, CHNH), 3.28 (1H, dd, J

= 14.2, 4.6 Hz, CH2CH), 3.04 (1H, dd, J = 14.2, 10.2 Hz, CH2CH), 2.73 (4H, s, 2 x CH2) ppm.

13C-NMR (67.5 MHz, ACETONE-D6) δ: 170.2 (2 x CO), 167.2, 157.6 (q,

2JCF = 38.0 Hz),

157.3, 131.2 (2 x CH), 126.9, 116.6 (q, 1JCF = 287.2 Hz), 116.2 (2 x CH), 53.2, 36.3, 26.2 (2 x

CH2) ppm. HRMS-ESI (m/z) [M+Na]+ calcd for C15H13F3N2O6Na 397.0623, found 397.0625.

(R)-2,5-dioxopyrrolidin-1-yl 3-(4-hydroxyphenyl)-2-(2,2,2-

trifluoroacetamido)propanoate (TFA-D-Tyr-OSu, D-99): Yellowish amorphous mass. [α]D

= +29 (c 1.0, MeOH). IR (neat) : 3323, 1820, 1788, 1732 cm-1

. 1H-NMR (270 MHz,

ACETONE-D6) δ: 8.87 (1H, d, J = 8.6 Hz, NH), 8.15 (1H, br s, OH), 7.10 (2H, d, J = 8.6 Hz,

Ar-H), 6.67 (2H, d, J = 8.6 Hz, Ar-H), 5.03 (1H, td, J = 9.9, 4.8 Hz, CHNH), 3.28 (1H, dd, J

= 14.3, 4.8 Hz, CH2CH), 3.04 (1H, dd, J = 14.2, 9.9 Hz, CH2CH), 2.74 (4H, s, 2 x CH2) ppm.

13C-NMR (67.5 MHz, ACETONE-D6) δ: 170.1 (2 x CO), 167.2, 157.6 (q,

2JCF = 37.4 Hz),

129

157.4, 131.2 (2 x CH), 127.0, 116.7 (q, J = 287.7 Hz), 116.2 (2 x CH), 53.3, 36.3, 26.2 (2 x

CH2) ppm. HRMS-ESI (m/z) [M+Na]+ calcd for C15H13F3N2O6Na 397.0623, found 397.0636.

3.3.4 General procedure for the preparation of TFA-protected -amino aryl-ketones

TFA-α-amino acid-OSu (0.1–0.5 mmol) was suspended in arenes or N-heterocycles

(300 equiv.). Into the suspension, pulverized AlCl3 was added and then stirred at a

temperature of 70 oC. The reaction was monitored by the consumption of starting material on

TLC. The solvent was reduced by rotary evaporation, then the mixture was poured into an

ethyl acetate-H2O two-phase system. The organic layer was washed with H2O, sat. NaCl,

dried over MgSO4, and then evaporated. The crude product was purified by silica column

chromatography (ethyl acetate/hexane 1:3 or and diethyl ether/hexane 1:5).

2,2,2-Trifluoro-N-((2S,3S)-3-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-L-Ile-

Ph, L-35): Colorless needles. [α]D = +70 (c 2.0, CHCl3). IR (neat) ν: 3317, 3073, 2972, 1722,

1694 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.97 (d, J = 7.4 Hz, 2H, Ar-H), 7.66 (t, J = 7.4 Hz,

1H, Ar-H), 7.53 (t, J = 7.4 Hz, 2H, Ar-H), 5.60 (dd, J = 8.6, 4.3 Hz, 1H, CHNH), 2.10–1.95

(m, 1H, CHCH3), 1.40–1.25 (m, 1H, CH2CH3), 1.12–0.95 (m, 4H, overlap CH2CH3 and

CHCH3), 0.82 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.5,

157.1 (q, 2JCF = 37.4 Hz), 134.7, 134.3, 129.0 (2 x CH), 128.7 (2 x CH), 115.9 (q,

1JCF =

288.1 Hz), 58.4, 38.7, 23.7, 16.2, 11.4 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C14H17F3NO2 288.1211, found 288.1212.

2,2,2-Trifluoro-N-((2R,3R)-3-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-D-Ile-

Ph, D-35): Colorless needles. [α]D = –70 (c 2.0, CHCl3). IR (neat) ν: 3337, 3069, 2969, 1721,

1699 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.6 Hz, 2H, Ar-H), 7.66 (t, J = 7.6 Hz,

1H, Ar-H), 7.53 (t, J = 7.6 Hz, 2H, Ar-H), 5.60 (dd, J = 8.6, 4.0 Hz, 1H, CHNH), 2.10–1.95

(m, 1H, CHCH3), 1.40–1.23 (m, 1H, CH2CH3), 1.12–0.95 (m, 4H, overlap CH2CH3 and

CHCH3), 0.82 (t, J = 7.3 Hz, 3H, CH2CH3) ppm.13

C-NMR (67.5 MHz, CDCl3) δ: 197.5,

157.1 (q, 2JCF = 37.2 Hz), 134.7, 134.3, 129.0 (2 x CH), 128.7 (2 x CH), 115.9 (q,

1JCF =

288.3 Hz), 58.4, 38.7, 23.7, 16.2, 11.4 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C14H17F3NO2 288.1211, found 288.1215.

2,2,2-Trifluoro-N-((2S,3S)-3-methyl-1-oxo-1-(p-tolyl)pentan-2-yl)acetamide (TFA-L-Ile-

Ph(4-Me), L-45): Colorless needles. [α]D = +83 (c 1.0, CHCl3). IR (neat) ν: 3302, 3023, 2925,

1700 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.87 (d, J = 8.2 Hz, 2H, Ar-H), 7.32 (d, J = 8.6 Hz,

2H, Ar-H), 5.56 (dd, J = 8.6, 4.3 Hz, 1H, CHNH), 2.45 (s, 3H, CH3), 2.06–1.95 (m, 1H,

CHCH3), 1.39–1.26 (m, 1H, CH2CH3), 1.11–0.94 (m, 4H, overlap CH2CH3 and CHCH3), 0.82

(t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.0, 157.1 (q, 2JCF =

130

37.2 Hz), 145.5, 132.1 (2 x CH), 129.7 (2 x CH), 128.8, 115.9 (q, 1JCF = 287.9 Hz), 58.3, 38.9,

23.7, 21.8, 16.2, 11.4 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C15H19F3NO2 302.1368

found 302.1353.

2,2,2-Trifluoro-N-((2S,3S)-1-(4-methoxyphenyl)-3-methyl-1-oxopentan-2-yl)acetamide

(TFA-L-Ile-Ph(4-OMe), L-47): Colorless oil. [α]D = +66 (c 1.0, CHCl3). IR (neat) ν: 3320,

3079, 2934, 1726, 1675 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.96 (d, J = 8.9 Hz, 2H, Ar-H),

6.99 (d, J = 8.9 Hz, 2H, Ar-H), 5.53 (dd, J = 8.7, 4.5 Hz, 1H, CHNH), 3.90 (s, 3H, OCH3),

2.08-1.93 (m, 1H, CHCH3), 1.42-1.20 (m, 1H, CH2CH3), 1.10-0.95 (m, 4H, overlap CH2CH3

and CHCH3), 0.82 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 195.6,

164.5, 157.0 (q, 2JCF = 37.2 Hz), 131.1 (2 x CH), 127.5, 115.9 (q,

1JCF = 288.6 Hz), 114.2 (2 x

CH), 58.0, 55.6, 39.0, 23.7, 16.2, 11.4 ppm. HRMS-ESI (m/z) [M + Na]+ calcd for

C15H18F3NO3Na 340.1136 found 340.1145.

N-((2S,3S)-1-(3,4-Dimethylphenyl)-3-methyl-1-oxopentan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Ile-Ph(3,4-Me), L-49): Colorless needles. [α]D = +82 (c 1.0, CHCl3). IR (neat) ν:

3343, 3075, 2979, 1742, 1691 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.71 (d, J = 11.2 Hz, 2H,

Ar-H), 7.29 (s, 1H, Ar-H), 5.56 (dd, J = 8.7, 4.1 Hz, 1H, CHNH), 2.35 (s, 6H, 2 x CH3), 2.06–

1.95 (m, 1H, CHCH3), 1.40–1.26 (m, 1H, CH2CH3), 1.08–0.93 (m, 4H, overlap CH2CH3 and

CHCH3), 0.81 (t, J = 7.4 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.2,

157.1 (q, 2JCF = 37.2 Hz), 144.3, 137.6, 132.5, 130.2, 129.7, 126.5, 115.9 (q,

1JCF = 287.9 Hz),

58.3, 38.9, 23.7, 20.1, 19.8, 16.2, 11.4 ppm. HRMS-ESI (m/z) [M + H]+ calcd for

C16H21F3NO2 316.1524, found 316.1506.

N-((2S,3S)-1-(2,4-Dimethylphenyl)-3-methyl-1-oxopentan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Ile-Ph(2,4-Me), L-50): Colorless needles. [α]D = +49 (c 0.25, CHCl3). IR (neat) ν:

3294, 3097, 2969, 1714, 1685 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.63 (d, J = 8.6 Hz, 1H,

Ar-H), 7.14-7.02 (m, 2H, Ar-H), 5.49 (dd, J = 8.4, 4.1 Hz, 1H, CHNH), 2.49 (s, 3H, CH3),

2.38 (s, 3H, CH3), 1.99–1.87 (m, 1H, CHCH3), 1.32–1.19 (m, 1H, CH2CH3), 1.07–0.87 (m,

4H, overlap CH2CH3 and CHCH3), 0.81 (t, J = 7.3 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 199.9, 157.1 (q, 2JCF = 37.4 Hz), 143.2, 139.5, 133.3, 132.2, 129.4, 126.6,

115.9 (q, 1JCF = 288.3 Hz), 59.8, 38.5, 24.1, 21.2, 21.0, 15.9, 11.2 ppm. HRMS-ESI (m/z) [M

+ H]+ calcd for C16H21F3NO2 316.1524, found 316.1526.

N-((2S,3S)-1-(2,5-Dimethylphenyl)-3-methyl-1-oxopentan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Ile-Ph(2,5-Me), L-51): Colorless needles. [α]D = +68 (c 1.0, CHCl3). IR (neat) ν:

3302, 3024, 2934, 1714, 1686, 1567 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.5 (s, 1H, Ar-H),

7.3 (d, J = 7.6 Hz, 1H, Ar-H), 7.2 (d, J = 7.6 Hz, 1H, Ar-H), 5.5 (dd, J = 8.6, 4.0 Hz, 1H,

CHNH), 2.4 (s, 3H, CH3), 2.4 (s, 3H, CH3), 2.0–1.8 (m, 1H, CHCH3), 1.4–1.2 (m, 1H,

131

CH2CH3), 1.1–0.9 (m, 3H overlap CH2CH3 and CHCH3), 0.8 (t, J = 7.3 Hz, 3H, CH2CH3)

ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 200.7, 157.1 (q, 2JCF = 37.2 Hz), 135.9, 135.6, 135.0,

133.3, 132.3, 129.3, 115.9 (q, 1JCF = 287.9 Hz), 60.1, 38.4, 24.1, 20.7, 20.3, 15.9, 11.3 ppm.

HRMS-ESI (m/z) [M + H]+ calcd for C16H21F3NO2 316.1524, found 316.1530.

2,2,2-Trifluoro-N-((2S,3R)-3-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-L-allo-

Ile-Ph, L-39): Colorless needles. [α]D = +79 (c 2.0, CHCl3). IR (neat) ν: 3335, 3068, 2971,

1741, 1693 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.6 Hz, 2H, Ar-H), 7.65 (t, J =

7.6 Hz, 1H, Ar-H), 7.53 (t, J = 7.6 Hz, 2H, Ar-H), 5.74 (dd, J = 8.9, 3.0 Hz, 1H, CHNH),

2.09–1.94 (m, 1H, CHCH3), 1.62–1.49 (m, 1H, CH2CH3), 1.38–1.21 (m, 1H, CH2CH3), 1.06

(t, J = 7.3 Hz, 3H, CH2CH3), 0.76 (d, J = 6.9 Hz, 3H, CHCH3) ppm. 13

C NMR (67.5 MHz,

CDCl3) δ: 197.1, 157.3 (q, 2JCF = 37.1 Hz), 134.4, 134.0, 129.1 (2 x CH), 128.7 (2 x CH),

115.9 (q, 1JCF = 287.7 Hz), 57.1, 38.7, 27.3, 13.4, 12.0 ppm. HRMS-ESI (m/z) [M + Na]

+

calcd for C14H16F3NO2Na 310.1031, found 310.1039.

2,2,2-Trifluoro-N-((2R,3S)-3-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-D-allo-

Ile-Ph, D-39): Colorless needles. [α]D = –79 (c 2.0, CHCl3). IR (neat) ν: 3331, 3067, 2969,

1738, 1692 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.4 Hz, 2H, Ar-H), 7.66 (t, J =

7.4 Hz, 1H, Ar-H), 7.53 (t, J = 7.4 Hz, 2H, Ar-H), 5.74 (dd, J = 8.7, 2.8 Hz, 1H, CHNH),

2.07–1.94 (m, 1H, CHCH3), 1.66–1.48 (m, 1H, CH2CH3), 1.38–1.21 (m, 1H, CH2CH3), 1.06

(t, J = 7.3 Hz, 3H, CH2CH3), 0.76 (d, J = 6.9 Hz, 3H, CHCH3) ppm. 13

C-NMR (67.5 MHz,

CDCl3) δ: 197.1, 157.3 (q, 2JCF = 37.4 Hz), 134.4, 134.1, 129.1 (2 x CH), 128.7 (2 x CH),

115.9 (q, 1JCF = 287.9 Hz), 57.1, 38.7, 27.3, 13.4, 12.0 ppm. HRMS-ESI (m/z) [M + Na]

+

calcd for C14H16F3NO2Na 310.1031, found 310.1040.

2,2,2-Trifluoro-N-(2-oxo-2-phenylethyl)acetamide (TFA-Gly-Ph, 54): 32

Colorless oil. IR

(neat) ν: 3327, 3103, 2927, 1733, 1703 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.99 (d, J = 7.4

Hz, 2H, Ar-H), 7.68 (t, J = 7.4 Hz, 1H, Ar-H), 7.54 (t, J = 7.4 Hz, 2H, Ar-H), 4.83 (d, J = 4.3

Hz, 2H, CH2NH) ppm. 13

C NMR (67.5 MHz, CDCl3) δ: 192.1, 157.1 (q, 2JCF = 37.6 Hz),

134.6, 133.6, 129.1 (2 x CH), 127.9 (2 x CH), 115.7 (q, 1JCF = 287.3 Hz), 46.1 ppm. HRMS-

ESI (m/z) [M + H]+ calcd for C10H9F3NO2 232.0585, found 232.0595.

(S)-2,2,2-trifluoro-N-(1-oxo-1-phenylpropan-2-yl)acetamide (TFA-L-Ala-Ph, L-58)

25,30,110: Colorless oil. = −7.0 (c 1.0, CHCl3). Lit.

110 [α]D = −8.6 (c 0.17, CHCl3). IR (neat) ν:

3331, 3070, 2991, 1738, 1701 cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.99 (d, J = 7.4 Hz, 2H,

Ar-H), 7.67 (t, J = 7.4 Hz, 1H, Ar-H), 7.54 (t, J = 7.4 Hz, 2H, Ar-H), 5.60–5.50 (m, 1H,

CHCH3), 1.53 (d, J = 7.3 Hz, 3H, CHCH3) ppm.13

C NMR (67.5 MHz, CDCl3) δ: 197.0, 156.5

(q, 2JCF = 37.6 Hz), 134.5, 132.9, 129.1 (2 x CH), 128.8 (2 x CH), 115.7 (q,

1JCF = 287.3 Hz),

50.8, 19.2 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C11H11F3NO2 246.0742, found 246.0748.

132

(R)-2,2,2-Trifluoro-N-(1-oxo-1-phenylpropan-2-yl)acetamide (TFA-D-Ala-Ph, D-58):

Colorless oil. [α]D = +7.0 (c 1.0, CHCl3). IR (neat) ν: 3337, 3091, 2948, 1725, 1700 cm−1

. 1H-

NMR (270 MHz, CDCl3) δ: 7.99 (d, J = 7.3 Hz, 2H, Ar-H), 7.67 (t, J = 7.4 Hz, 1H, Ar-H),

7.54 (t, J = 7.4 Hz, 2H, Ar-H), 5.60–5.49 (m, 1H, CHCH3), 1.53 (d, J = 6.9 Hz, 3H, CHCH3)

ppm. 13

C NMR (67.5 MHz, CDCl3) δ: 197.0, 156.5 (q, 2JCF = 37.6 Hz), 134.5, 132.9, 129.1 (2

x CH), 128.8 (2 x CH), 115.7 (q, 1JCF = 287.7 Hz), 50.8, 19.2 ppm. HRMS-ESI (m/z) [M +

H]+ calcd for C11H11F3NO2 246.0742, found 246.0750.

(S)-2,2,2-Trifluoro-N-(3-methyl-1-oxo-1-phenylbutan-2-yl)acetamide (TFA-L-Val-Ph, L-

62):25

Colorless oil. [α]D = +83 (c 1.0, CHCl3). IR (neat) ν: 3347, 3071, 2972, 1728, 1679

cm−1

. (270 MHz, CDCl3) δ: 7.99 (d, J = 7.7 Hz, 2H, Ar-H), 7.66 (t, J = 7.7 Hz, 1H, Ar-H),

7.53 (t, J = 7.7 Hz, 2H, Ar-H), 7.32 (br s, 1H, NH), 5.61 (dd, J = 8.6, 4.0 Hz, 1H, CHNH),

2.39–2.23 (m, 1H, CHCH3), 1.07 (d, J = 6.9 Hz, 3H, CHCH3), 0.80 (d, J = 6.9 Hz, 3H,

CHCH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.2, 157.3 (q, 2JCF = 37.6 Hz), 134.4, 129.1

(2 x CH), 128.7 (2 x CH), 115.9 (q, 1JCF = 287.7 Hz), 58.6, 32.2, 20.0, 16.4 ppm. HRMS-ESI

(m/z) [M + H]+ calcd for C13H15F3NO2 274.1055, found 274.1057.

(R)-2,2,2-Trifluoro-N-(3-methyl-1-oxo-1-phenylbutan-2-yl)acetamide (TFA-D-Val-Ph, D-

62): Colorless oil. [α]D = −83 (c 1.0, CHCl3). IR (neat) ν: 3344, 3070, 2972, 1720, 1672 cm−1

.

1H-NMR (270 MHz, CDCl3) δ: 7.99 (d, J = 7.3 Hz, 2H, Ar-H), 7.66 (t, J = 7.3 Hz, 1H, Ar-H),

7.53 (t, J = 7.3 Hz, 2H, Ar-H), 7.30 (br s, 1H, NH), 5.61 (dd, J = 8.7, 3.8 Hz, 1H, CHNH),

2.39–2.22 (m, 1H, CHCH3), 1.07 (d, J = 6.9 Hz, 3H, CHCH3), 0.79 (d, J = 6.9 Hz, 3H,

CHCH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.2, 157.3 (q, 2JCF = 36.7 Hz), 134.4, 129.1

(2 x CH), 128.7 (2 x CH), 115.9 (q, 1JCF = 287.7 Hz), 58.6, 32.1, 19.9, 16.3 ppm. HRMS-ESI

(m/z) [M + H]+ calcd for C13H15F3NO2 274.1055, found 274.1057.

(S)-2,2,2-Trifluoro-N-(4-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-L-Leu-Ph,

L-66): Colorless oil. [α]D = +26 (c 2.0, CHCl3). IR (neat) ν: 3334, 3092, 2963, 1726, 1683

cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.3 Hz, 2H, Ar-H), 7.66 (t, J = 7.4 Hz, 1H,

Ar-H), 7.54 (t, J = 7.6 Hz, 2H, Ar-H), 5.68 (td, J = 8.9, 2.6 Hz, 1H, CHNH), 1.79–1.49 (m,

3H, CH2CH), 1.10 (d, J = 5.9 Hz, 3H, CHCH3), 0.89 (d, J = 6.3 Hz, 3H, CHCH3) ppm. 13

C-

NMR (67.5 MHz, CDCl3) δ: 197.4, 156.9 (q, 2JCF = 37.4 Hz), 134.4, 133.6, 129.1 (2 x CH),

128.7 (2 x CH), 115.8 (q, 1JCF = 287.7 Hz), 52.9, 42.7, 25.1, 23.3, 21.7 ppm. HRMS-ESI

(m/z) [M + H]+ calcd for C14H17F3NO2 288.1211, found 288.1214.

(R)-2,2,2-Trifluoro-N-(4-methyl-1-oxo-1-phenylpentan-2-yl)acetamide (TFA-D-Leu-Ph,

D-66): Colorless oil. [α]D = –26 (c 2.0, CHCl3). IR (neat) ν: 3335, 3094, 2931, 1731, 1685

cm−1

. 1H-NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.3 Hz, 2H, Ar-H), 7.66 (t, J = 7.3 Hz, 1H,

Ar-H), 7.54 (t, J = 7.3 Hz, 2H, Ar-H), 5.68 (td, J = 9.1, 2.5 Hz, 1H, CHNH), 1.79–1.53 (m,

133

3H, CH2CH), 1.10 (d, J = 5.9 Hz, 3H, CHCH3), 0.89 (d, J = 5.9 Hz, 3H, CHCH3) ppm. 13

C-

NMR (67.5 MHz, CDCl3) δ: 197.4, 156.9 (q, 2JCF =37.4 Hz), 134.4, 133.6, 129.1 129.1 (2 x

CH), 128.7 129.1 (2 x CH), 115.8 (q, 1JCF = 287.7 Hz), 52.9, 42.6, 25.1, 23.2, 21.7 ppm.

HRMS-ESI (m/z) [M + H]+ calcd for C14H17F3NO2 288.1211, found 288.1220.

(S)-2,2,2-Trifluoro-N-(1-oxo-1-phenylpentan-2-yl)acetamide (TFA-L-Nva-Ph, L-70):

Colorless oil. [α]D = +46 (c 1.0, CHCl3). IR (neat) ν: 3341, 3074, 2979, 1733 cm−1

. 1H-NMR

(270 MHz, CDCl3) δ: 7.98 (d, J = 7.3 Hz, 2H, Ar-H), 7.67 (t, J = 7.3 Hz, 1H, Ar-H), 7.54 (t, J

= 7.3 Hz, 2H, Ar-H), 7.44 (br s, 1H, NH), 5.62 (td, J = 7.4, 4.5 Hz, 1H, CHNH), 2.08–1.95 (m,

1H, CHCH2), 1.76–1.62 (m, 1H, CHCH2), 1.48–1.17 (m, 2H, CH2CH3) 0.90 (t, J = 7.3 Hz, 3H,

CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.0, 156.8 (q, 2JCF = 37.1 Hz), 134.5,

133.6, 129.1 (2 x CH), 128.7 (2 x CH), 115.8 (q, 1JCF = 286.8 Hz), 54.4, 35.2, 18.0, 13.7 ppm.

HRMS-ESI (m/z) [M + H]+ calcd for C13H15F3NO2 274.1055, found 274.1064.

(R)-2,2,2-Trifluoro-N-(1-oxo-1-phenylpentan-2-yl)acetamide (TFA-D-Nva-Ph, D-70):

Colorless oil. [α]D = −46 (c 1.0, CHCl3). IR (neat) ν: 3339, 3073, 2977, 1732 cm−1

. 1H-NMR

(270 MHz, CDCl3) δ: 7.99 (d, J = 7.4 Hz, 2H, Ar-H), 7.67 (t, J = 7.4 Hz, 1H, Ar-H), 7.54 (t, J

= 7.4 Hz, 2H, Ar-H), 7.45 (br s, 1H, NH), 5.62 (td, J = 7.3, 4.4 Hz, 1H, CHNH), 2.08–1.95 (m,

1H, CHCH2), 1.76–1.62 (m, 1H, CHCH2), 1.48–1.13 (m, 2H, CH2CH3), 0.90 (t, J = 7.3 Hz,

3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.0, 156.8 (q, 2JCF = 37.4 Hz), 134.5,

133.6, 129.1 (2 x CH), 128.7 (2 x CH), 115.8 (q, 1JCF = 288.3 Hz), 54.4, 35.2, 18.1, 13.7 ppm.

HRMS-ESI (m/z) [M + H]+ calcd for C13H15F3NO2 274.1055, found 274.1058.

(S)-2,2,2-Trifluoro-N-(1-oxo-1-phenylhexan-2-yl)acetamide (TFA-L-Nle-Ph, L-74):

Colorless oil. [α]D = +60 (c 0.5, CHCl3). IR (neat) ν: 3326, 3068, 2960, 1728, 1687 cm−1

. 1H-

NMR (270 MHz, CDCl3) δ: 7.98 (d, J = 7.4 Hz, 2H, Ar-H), 7.66 (t, J = 7.4 Hz, 1H, Ar-H),

7.53 (t, J = 7.4 Hz, 2H, Ar-H), 7.46 (br s, 1H, NH), 5.61 (td, J = 7.3, 4.6 Hz, 1H, CHNH),

2.12–1.98 (m, 1H, CHCH2), 1.77–1.63 (m, 1H, CHCH2), 1.41–1.15 (m, 4H, 2 x CH2), 0.83 (t,

J = 6.9 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.0, 156.8 (q, 2JCF = 37.4

Hz), 134.4, 133.6, 129.0 (2 x CH), 128.6 (2 x CH), 115.8 (q, 1JCF = 287.7 Hz), 54.5, 32.7,

26.7, 22.2, 13.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C14H17F3NO2 288.1211, found

288.1224.

(R)-2,2,2-Trifluoro-N-(1-oxo-1-phenylhexan-2-yl)acetamide (TFA-D-Nle-Ph, D-74):

Colorless oil. [α]D = −60 (c 0.5, CHCl3). IR (neat) ν: 3327, 3069, 2961, 1732, 1691 cm−1

. 1H-

NMR (270 MHz, CDCl3) δ: 7.99 (d, J = 7.3 Hz, 2H, Ar-H), 7.67 (t, J = 7.3 Hz, 1H, Ar-H),

7.54 (t, J = 7.3 Hz, 2H, Ar-H), 7.47 (br s, 1H, NH), 5.61 (td, J = 7.3, 4.6 Hz, 1H, CHNH),

2.12–1.98 (m, 1H, CHCH2), 1.78–1.63 (m, 1H, CHCH2), 1.39–1.14 (m, 4H, 2 x CH2), 0.83 (t,

J = 6.9 Hz, 3H, CH2CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 197.0, 156.8 (q, 2JCF = 37.4

134

Hz), 134.4, 133.6, 129.1 (2 x CH), 128.6 (2 x CH), 115.8 (q, 1JCF = 287.7 Hz), 54.5, 32.7,

26.7, 22.2, 13.6 ppm. HRMS-ESI (m/z) [M + H]+ calcd for C14H17F3NO2 288.1211, found

288.1216.

(S)-1-(2-benzoylpyrrolidin-1-yl)-2,2,2-trifluoroethanone (TFA-L-Pro-Ph, L-78):25,32,99

Colorless needles. [α]D = –81 (c 1.0, CHCl3). IR (neat) : 3361, 3136, 1776, 1711, 1695 cm-1

.

1H-NMR (270 MHz, CDCl3) δ: 7.99 (2H, d, J = 7.9 Hz, Ar-H), 7.62 (1H, t, J = 7.9 Hz, Ar-H),

7.50 (2H, t, J = 7.9 Hz, Ar-H), 5.57 (1H, dd, J = 9.2, 4.0 Hz, CH2CH), 3.99–3.77 (2H, m,

CH2), 2.42–2.29 (2H, m, CH2), 2.18–1.98 (2H, m, CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3)

δ: [195.6 & 195.3], 155.5 (q, 2JCF = 37.4 Hz), [134.4 & 134.0], [133.7 & 133.3], 129.0, 128.8,

128.5, 128.4, 116.3 (q, 1JCF = 287.2 Hz), [62.6 & 62.1], [48.3 & 47.3], [31.3 & 28.5], [24.8 &

20.8] ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd for C13H13F3NO2 272.0898,

found 272.0898.

(R)-1-(2-benzoylpyrrolidin-1-yl)-2,2,2-trifluoroethanone (TFA-D-Pro-Ph, D-78):

Colorless needles. [α]D = +81 (c 1.0, CHCl3). IR (neat) : 3362, 3135, 1773, 1711, 1696 cm-1

.

1H-NMR (270 MHz, CDCl3) δ: 7.99 (2H, d, J = 7.6 Hz, Ar-H), 7.62 (1H, t, J = 7.6 Hz, Ar-H),

7.50 (2H, t, J = 7.6 Hz, Ar-H), 5.58 (1H, dd, J = 9.1, 3.8 Hz, CH2CH), 4.00–3.77 (2H, m,

CH2), 2.41–2.30 (1H, m, CH2), 2.15–1.97 (2H, m, CH2) ppm. 13

C-NMR (67.5 MHz, CDCl3)

δ: [195.6 & 195.3], 155.5 (q, 2JCF = 37.4 Hz), [134.4 & 134.0], [133.7 & 133.3], 129.0, 128.8,

128.5, 128.4, 116.3 (q, 1JCF = 287.2 Hz), [62.6 & 62.1], [48.3 & 47.4], [31.2 & 28.5], [24.7 &

20.8] ppm (cis- and trans- mix). HRMS-ESI (m/z) [M+H]+ calcd for C13H13F3NO2 272.0898,

found 272.0916.

(S)-2,2,2-trifluoro-N-(1-oxo-1,3-diphenylpropan-2-yl)acetamide (TFA-L-Phe-Ph, L-83):

Colorless needles; [α]D = +14.0 (c 1.0, MeOH). IR (neat) : 3326, 3065, 3032, 1728, 1686

cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.96 (2H, d, J = 7.4 Hz, Ar-H), 7.66 (1H, t, J = 7.4 Hz,

Ar-H), 7.52 (2H, t, J = 7.4 Hz, Ar-H), 7.31 (1H, br d, J = 6.3 Hz, NH), 7.23–7.20 (3H, m, Ar-

H), 6.92 (2H, dd, J = 6.4, 2.8 Hz, Ar-H), 5.82 (1H, q, J = 6.3, 6.3, 6.3 Hz, CHNH), 3.37 (1H,

dd, J = 14.2, 6.3 Hz, CH2CH), 3.12 (1H, dd, J = 14.2, 6.3 Hz, CH2CH) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 195.9, 156.5 (q, 2JCF = 37.6 Hz), 134.4, 134.2, 133.8, 129.4 (2 x CH), 129.1

(2 x CH), 128.8 (2 x CH), 128.5 (2 x CH), 127.4, 115.7 (q, 1JCF = 287.7 Hz), 55.3, 38.2 ppm.

HRMS-ESI (m/z) [M+H]+ calcd for C17H15F3NO2 322.1055, found 322.1065.

(R)-2,2,2-trifluoro-N-(1-oxo-1,3-diphenylpropan-2-yl)acetamide (TFA-D-Phe-Ph, D-83):

Colorless needles; [α]D = –14.0 (c 1.0, MeOH). IR (neat) : 3326, 3066, 3032, 1720, 1684

cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.96 (2H, d, J = 7.3 Hz, Ar-H), 7.65 (1H, t, J = 7.3 Hz,

Ar-H), 7.52 (2H, t, J = 7.3 Hz, Ar-H), 7.40 (1H, br d J = 5.9 Hz, NH), 7.23–7.20 (3H, m, Ar-

135

H), 6.92 (2H, dd, J = 6.3, 3.0 Hz, Ar-H), 5.82 (1H, q, J = 5.9, 5.9, 5.9 Hz, CHNH), 3.37 (1H,

dd, J = 14.2, 5.9 Hz, CH2CH), 3.11 (1H, dd, J = 14.2, 5.9 Hz, CH2CH) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 195.9, 156.5 (q, 2JCF = 37.4 Hz), 134.4, 134.2, 133.8, 129.4 (2 x CH), 129.1

(2 x CH), 128.7 (2 x CH), 128.5 (2 x CH), 127.4, 115.6 (q, 1JCF = 287.7 Hz), 55.3, 38.2 ppm.

HRMS-ESI (m/z) [M+H]+ calcd for C17H15F3NO2 322.1055, found 322.1065.

(S)-2,2,2-trifluoro-N-(1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (TFA-L-cPhe, L-84):30

Colorless amorphous mass; [α]D = –1.4 (c 1.0, acetone); –3.1 (c 1.6, DMF); Lit. 30

[α]D = –3.2

(c 1.6, DMF). IR (neat) : 3301, 3108, 2954, 1733, 1700 cm-1

. 1H-NMR (270 MHz,

ACETONE-D6) δ: 8.97 (1H, br d, J = 5.4 Hz, NH), 7.75–7.68 (2H, m, Ar-H), 7.58 (1H, d, J =

7.4 Hz, Ar-H), 7.47 (1H, t, J = 7.4 Hz, Ar-H), 4.74 (1H, ddd, J = 8.6, 5.4, 5.4 Hz, CHNH),

3.68 (1H, dd, J = 16.8, 8.6 Hz, CH2CH), 3.20 (1H, dd, J = 16.8, 5.4 Hz, CH2CH) ppm. 13

C-

NMR (67.5 MHz, ACETONE-D6) δ: 201.2, 157.7 (q, 2JCF = 36.9 Hz), 152.1, 136.3, 135.9,

128.7, 127.6, 124.4, 117.0 (q, 1JCF = 287.7 Hz), 56.0, 33.2 ppm. HRMS-ESI (m/z) [M+H]

+

calcd for C11H9F3NO2 244.0585, found 244.0586.

(R)-2,2,2-trifluoro-N-(1-oxo-2,3-dihydro-1H-inden-2-yl)acetamide (TFA-D-cPhe, D-84):

Colorless amorphous mass; [α]D = +1.6 (c 1.0, acetone); +3.1 (c 1.6, DMF). IR (neat) : 3300,

3109, 2955, 1733, 1699 cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.94 (1H, br d, J = 5.3

Hz, NH), 7.75–7.68 (2H, m, Ar-H), 7.58 (1H, d, J = 7.4 Hz, Ar-H), 7.47 (1H, t, J = 7.4 Hz,

Ar-H), 4.74 (1H, ddd, J = 8.4, 5.3, 5.3 Hz, CHNH), 3.68 (1H, dd, J = 16.8, 8.4 Hz, CH2CH),

3.20 (1H, dd, J = 16.8, 5.3 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz, ACETONE-D6) δ: 201.2,

157.7 (q, 2JCF = 36.3 Hz), 152.1, 136.3, 135.9, 128.7, 127.6, 124.4, 117.0 (q,

1JCF = 287.7 Hz),

56.0, 33.2 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C11H9F3NO2 244.0585, found 244.0588.

(S)-2,2,2-trifluoro-N-(1-oxo-3-phenyl-1-(p-tolyl)propan-2-yl)acetamide (TFA-L-Phe-

Ph(4-Me), L-85): Colorless needles; [α]D = +16 (c 1.0, MeOH). IR (neat) : 3304, 3091,

3034, 2932, 1708, 1680 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.87 (2H, d, J = 8.2 Hz, Ar-H),

7.33 (4H, d, J = 8.2 Hz, Ar-H), 7.23–7.20 (1H, m, Ar-H), 6.92–6.88 (2H, m, Ar-H), 5.77 (1H,

ddd, J = 5.9, 5.9, 5.9 Hz, CHNH), 3.38 (1H, dd, J = 14.2, 5.9 Hz, CH2CH), 3.11 (1H, dd, J =

14.2, 5.9 Hz, CH2CH), 2.46 (3H, s, CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 195.4, 156.4

(q, 2JCF = 37.4 Hz), 145.6, 134.4, 131.2, 129.7 (2 x CH), 129.4 (2 x CH), 128.8 (2 x CH),

128.4 (2 x CH), 127.3, 115.6 (q, 1JCF = 287.7 Hz), 55.1, 38.2, 21.6 ppm. HRMS-ESI (m/z)

[M+H]+ calcd for C18H17F3NO2 336.1211, found 336.1180.

(R)-2,2,2-trifluoro-N-(1-oxo-3-phenyl-1-(p-tolyl)propan-2-yl)acetamide (TFA-D-Phe-

Ph(4-Me), D-85): Colorless needles; [α]D = –16 (c 1.0, MeOH). IR (neat) : 3301, 3090,

3034, 2931, 1708, 1680 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.87 (2H, d, J = 8.2 Hz, Ar-H),

136

7.33 (4H, d, J = 8.2 Hz, Ar-H), 7.23–7.20 (1H, m, Ar-H), 6.93–6.89 (2H, m, Ar-H), 5.78 (1H,

ddd, J = 5.8, 5.8, 5.8 Hz, CHNH), 3.37 (1H, dd, J = 14.0, 5.8 Hz, CH2CH), 3.11 (1H, dd, J =

14.0, 5.8 Hz, CH2CH), 2.45 (3H, s, CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 195.3, 156.5

(q, 2JCF = 38.0 Hz), 145.7, 134.3, 131.2, 129.8 (2 x CH), 129.4 (2 x CH), 128.9 (2 x CH),

128.5 (2 x CH), 127.4, 115.7 (q, 1JCF = 287.7 Hz), 55.1, 38.3, 21.7 ppm. HRMS-ESI (m/z)

[M+H]+ calcd for C18H17F3NO2 336.1211, found 336.1186.

(S)-2,2,2-trifluoro-N-(1-(4-methoxyphenyl)-1-oxo-3-phenylpropan-2-yl)acetamide (TFA-

L-Phe-Ph(4-OMe), L-86): Colorless amorphous mass; [α]D = +27 (c 1.0, MeOH). IR (neat)

: 3300, 3089, 3033, 2961, 2934, 1712, 1671 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.94 (2H,

d, J = 8.9 Hz, Ar-H), 7.40 (1H, br d, J = 5.6 Hz, NH), 7.23–7.20 (3H, m, Ar-H), 6.98 (2H, d, J

= 8.9 Hz, Ar-H), 6.95–6.91 (2H, m, Ar-H), 5.75 (1H, ddd, J = 5.6, 5.6, 5.6 Hz, CHNH), 3.90

(3H, s, OCH3), 3.35 (1H, dd, J = 14.0, 5.6 Hz, CH2CH), 3.11 (1H, dd, J = 14.0, 5.6 Hz,

CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 194.1, 164.6, 156.4 (q, 2JCF = 37.4 Hz), 134.4,

131.2 (2 x CH), 129.4 (2 x CH), 128.5 (2 x CH), 127.3, 126.6, 115.7 (q, 1JCF = 287.7 Hz),

114.3 (2 x CH), 55.6, 54.9, 38.6 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C18H17F3NO3

352.1161, found 352.1158.

(R)-2,2,2-trifluoro-N-(1-(4-methoxyphenyl)-1-oxo-3-phenylpropan-2-yl)acetamide (TFA-

D-Phe-Ph(4-OMe), D-86): Colorless amorphous mass; [α]D = –27 (c 1.0, MeOH). IR (neat)

: 3352, 3090, 3033, 2944, 2926, 1727, 1671 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.94 (2H,

d, J = 8.9 Hz, Ar-H), 7.37 (1H, br d, J = 5.6 Hz, NH), 7.23–7.20 (3H, m, Ar-H), 6.98 (2H, d, J

= 8.9 Hz, Ar-H), 6.94–6.90 (2H, m, Ar-H), 5.74 (1H, ddd, J = 5.6, 5.6, 5.6 Hz, CHNH), 3.91

(3H, s, OCH3), 3.35 (1H, dd, J = 14.0, 5.6 Hz, CH2CH), 3.11 (1H, dd, J = 14.0, 5.6 Hz,

CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 194.1, 164.5, 156.4 (q, 2JCF =37.6 Hz), 134.5,

131.1 (2 x CH), 129.3 (2 x CH), 128.3 (2 x CH), 127.2, 126.5, 115.7 (q, 1JCF = 287.3 Hz),

114.2 (2 x CH), 55.4, 54.8, 38.4 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C18H17F3NO3

352.1161, found 352.1169.

(S)-N-(1-(3,4-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Phe-Ph(3,4-Me), L-89): Colorless needles; [α]D = +16 (c 1.0, MeOH). IR (neat) :

3326, 3031, 2945, 2935, 1732, 1685 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.71 (2H, d, J = 6.9

Hz, Ar-H), 7.46 (1H, br d, J = 5.9 Hz, NH), 7.28–7.19 (4H, m, Ar-H), 6.93 (2H, d, J = 7.8 Hz,

Ar-H), 5.79 (1H, ddd, J = 4.9, 5.9, 5.9 Hz, CHNH), 3.35 (1H, dd, J = 13.8, 5.9 Hz, CH2CH),

3.10 (1H, dd, J = 13.8, 4.9 Hz, CH2CH), 2.33 (3H, s, CH3), 2.32 (3H, s, CH3) ppm. 13

C-NMR

(67.5 MHz, CDCl3) δ: 195.5, 156.4 (q, 2JCF = 37.4 Hz), 144.4, 137.6, 134.4, 131.5, 130.2,

129.8, 129.4 (2 x CH), 128.4 (2 x CH), 127.2, 126.5, 115.7 (q, 1JCF = 287.7 Hz), 55.1, 38.3,

20.0, 19.6 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C19H19F3NO2 350.1368, found 350.1356.

137

(R)-N-(1-(3,4-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-D-Phe-Ph(3,4-Me), D-89): Colorless needles; [α]D = –16 (c 1.0, MeOH). IR (neat) :

3329, 3031, 2946, 2925, 1735, 1687 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.71 (2H, d, J = 6.9

Hz, Ar-H), 7.45 (1H, br d, J = 5.9 Hz, NH), 7.28–7.19 (4H, m, Ar-H), 6.93 (2H, d, J = 7.8 Hz,

Ar-H), 5.79 (1H, ddd, J = 4.9, 5.9, 5.9 Hz, CHNH), 3.35 (1H, dd, J = 13.8, 5.9 Hz, CH2CH),

3.10 (1H, dd, J = 13.8, 4.9 Hz, CH2CH), 2.34 (3H, s, CH3), 2.32 (3H, s, CH3) ppm. 13

C-NMR

(67.5 MHz, CDCl3) δ: 195.5, 156.4 (q, 2JCF =37.2 Hz), 144.4, 137.6, 134.4, 131.5, 130.2,

129.8, 129.4 (2 x CH), 128.4 (2 x CH), 127.3, 126.5, 115.7 (q, 1JCF = 288.3 Hz), 55.1, 38.3,

20.0, 19.7 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C19H19F3NO2 350.1368, found 350.1362.

(S)-N-(1-(2,4-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Phe-Ph(2,4-Me), L-90): Colorless needles; [α]D = +11 (c 1.0, MeOH). IR (neat) :

3301, 3090, 3032, 2931, 1706, 1675 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.69 (1H, d, J = 8.6

Hz, Ar-H), 7.48 (1H, br d, J = 5.4 Hz, NH), 7.21–7.18 (3H, m, Ar-H), 7.15–7.11 (2H, m, Ar-

H), 6.87–6.83 (2H, m, Ar-H), 5.74 (1H, ddd, J = 5.4, 5.4, 5.4 Hz, CHNH), 3.32 (1H, dd, J =

14.0, 5.4 Hz, CH2CH), 3.04 (1H, dd, J = 14.0, 5.4 Hz, CH2CH), 2.40 (6H, s, 2 x CH3) ppm.

13C-NMR (67.5 MHz, CDCl3) δ: 197.3, 156.5 (q,

2JCF = 37.8 Hz), 144.0, 140.9, 134.4, 133.7,

130.5, 129.6, 129.3 (2 x CH), 128.5 (2 x CH), 127.3, 126.8, 115.7 (q, 1JCF = 287.7 Hz), 56.3,

37.8, 21.6, 21.5 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C19H19F3NO2 350.1368, found

350.1375.

(R)-N-(1-(2,4-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-D-Phe-Ph(2,4-Me), D-90): Colorless needles; [α]D = –11 (c 1.0, MeOH). IR (neat) :

3302, 3091, 3019, 2931, 1708, 1676 cm-1

. 1H-NMR (270 MHz, CDCl3) δ:

7.69 (1H, d, J = 8.9

Hz, Ar-H), 7.45 (1H, br d, J = 5.4 Hz, NH), 7.21–7.19 (3H, m, Ar-H), 7.16–7.12 (2H, m, Ar-

H), 6.86–6.83 (2H, m, Ar-H), 5.74 (1H, ddd, J = 5.4, 5.4, 5.4 Hz, CHNH), 3.32 (1H, dd, J =

14.2, 5.4 Hz, CH2CH), 3.05 (1H, dd, J = 14.2, 5.4 Hz, CH2CH), 2.40 (6H, s, 2 x CH3) ppm.

13C-NMR (67.5 MHz, CDCl3) δ: 197.3, 156.5 (q,

2JCF = 37.6 Hz), 144.0, 140.9, 134.4, 133.7,

130.5, 129.6, 129.3 (2 x CH), 128.5 (2 x CH), 127.3, 126.8, 115.7 (q, 1JCF = 287.3 Hz), 56.3,

37.8, 21.5, 21.5 ppm. HRMS-ESI (m/z) [M+H]+ calcd for C19H19F3NO2 350.1368, found

350.1371.

(S)-N-(1-(2,5-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-L-Phe-Ph(2,5-Me), L-91): Colorless needles; [α]D = +10 (c 1.0, MeOH). IR (neat) :

3303, 3033, 2927, 1708, 1679 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.45–7.38 (2H, m, Ar-H),

7.17–7.05 (4H, m, Ar-H), 6.80–6.74 (2H, m, Ar-H), 5.63 (1H, ddd, J = 5.6, 5.6, 5.6 Hz,

CHNH), 3.16 (1H, dd, J = 14.2, 5.6 Hz, CH2CH), 2.93 (1H, dd, J = 14.2, 5.6 Hz, CH2CH),

138

2.25 (3H, s, CH3), 2.23 (3H, s, CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 198.2, 156.5 (q,

2JCF = 37.6 Hz), 137.1, 135.7, 134.5, 133.6, 133.3, 132.5, 129.7, 129.2 (2 x CH), 128.4 (2 x

CH), 127.2, 115.7 (q, 1JCF = 287.0 Hz), 56.6, 37.6, 20.7, 20.7 ppm. HRMS-ESI (m/z) [M+H]

+

calcd for C19H19F3NO2 350.1368, found 350.1366.

(R)-N-(1-(2,5-dimethylphenyl)-1-oxo-3-phenylpropan-2-yl)-2,2,2-trifluoroacetamide

(TFA-D-Phe-Ph(2,5-Me), D-91): Colorless needles; [α]D = –10 (c 1.0, MeOH). IR (neat) :

3325, 3031, 2928, 1726, 1685 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.45–7.38 (2H, m, Ar-H),

7.16–7.05 (4H, m, Ar-H), 6.79–6.74 (2H, m, Ar-H), 5.63 (1H, ddd, J = 5.8, 5.8, 5.8 Hz,

CHNH), 3.16 (1H, dd, J = 14.2, 5.8 Hz, CH2CH), 2.93 (1H, dd, J = 14.2, 5.8 Hz, CH2CH),

2.25 (3H, s, CH3), 2.23 (3H, s, CH3) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 198.2, 156.5 (q,

2JCF = 37.2 Hz), 137.1, 135.6, 134.5, 133.6, 133.3, 132.4, 129.6, 129.2 (2 x CH), 128.4 (2 x

CH), 127.2, 115.7 (q, 1JCF = 286.8 Hz), 56.6, 37.6, 20.7, 20.7 ppm. HRMS-ESI (m/z) [M+H]

+

calcd for C19H19F3NO2 350.1368, found 350.1373.

(S)-2,2,2-trifluoro-N-(1-oxo-3-phenyl-1-(1H-pyrrol-2-yl)propan-2-yl)acetamide (TFA-L-

Phe-(2-Pyr), L-94):30

Colorless amorphous mass; [α]D = +40 (c 1.0, CHCl3). Lit.30

[α]D =

+44.3 (c 1.6, CHCl3). IR (neat) : 3308, 1701, 1654 cm-1

. 1H-NMR (270 MHz, CDCl3) δ:

9.49 (1H, br s, NH), 7.24–7.20 (3H, m, Ar-H), 7.13-7.11 (1H, m, Ar-H), 7.03–6.95 (3H, m,

Ar-H & CH), 6.34 (1H, dd, J = 6.4, 2.5 Hz, CH), 5.48 (1H, dd, J = 13.8, 5.9 Hz, CHNH), 3.35

(1H, dd, J = 13.8, 5.9 Hz, CH2CH), 3.17 (1H, dd, J = 13.8, 5.9 Hz, CH2CH) ppm. 13

C-NMR

(67.5 MHz, CDCl3) δ: 184.8, 156.4 (q, 2JCF = 37.4 Hz), 134.7, 129.4 (2 x CH), 129.0, 128.5

(2 x CH), 127.4, 126.6, 118.3, 115.7 (q, 1JCF = 287.7 Hz), 111.8, 55.3, 39.8 ppm. HRMS-ESI

(m/z) [M+Na]+ calcd for C15H13F3N2O2Na 333.0821, found 333.0824.

(R)-2,2,2-trifluoro-N-(1-oxo-3-phenyl-1-(1H-pyrrol-2-yl)propan-2-yl)acetamide (TFA-D-

Phe-(2-Pyr), D-94): Colorless amorphous mass; [α]D = –40 (c 1.0, CHCl3). IR (neat) : 3309,

1701, 1655 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 9.50 (1H, br s, NH), 7.24–7.20 (3H, m, Ar-

H), 7.13–7.10 (1H, m Ar-H), 7.02–6.98 (3H, m, Ar-H & CH), 6.34 (1H, dd, J = 6.4, 2.5 Hz,

CH), 5.48 (1H, dd, J = 13.8, 5.9 Hz, CHNH), 3.35 (1H, dd, J = 13.8, 5.9 Hz, CH2CH), 3.17

(1H, dd, J = 13.8, 5.9 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 184.8, 156.4 (q,

2JCF = 38.0 Hz), 134.7, 129.4 (2 x CH), 129.0, 128.5 (2 x CH), 127.4, 126.6, 118.3, 115.7 (q,

1JCF = 287.7 Hz), 111.8, 55.2, 39.8 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd for

C15H13F3N2O2Na 333.0821, found 333.0821.

(S)-2,2,2-trifluoro-N-(1-(1-methyl-1H-pyrrol-2-yl)-1-oxo-3-phenylpropan-2-yl)acetamide

(TFA-L-Phe-(2-(N-Me)Pyr), L-95): Yellowish amorphous mass; [α]D = +53 (c 1.0, CHCl3);

Lit. 30

[α]D = +43.6 (c 1.6, CHCl3). IR (neat) : 3289, 3123, 3088, 1724 cm-1

. 1H-NMR (270

139

MHz, CDCl3) δ: 7.36 (1H, br s, NH), 7.26–7.20 (4H, m, Ar-H), 7.04–6.99 (2H, m, Ar-H &

CH), 6.62 (2H, d, J = 2.0 Hz, CH), 5.32 (1H, dd, J = 13.8, 6.3 Hz, CHNH), 3.67 (3H, s, CH3),

3.31 (1H, dd, J = 13.8, 6.3 Hz, CH2CH), 3.15 (1H, dd, J = 13.8, 6.3 Hz, CH2CH) ppm. 13

C-

NMR (67.5 MHz, CDCl3) δ: 189.8, 156.4 (q, 2JCF = 37.4 Hz), 135.1, 129.5 (2 x CH), 128.3 (2

x CH), 128.0, 127.1, 124.1, 122.3, 115.7 (q, 1JCF = 287.7 Hz), 109.8, 56.2, 39.2, 36.7 ppm.

HRMS-ESI (m/z) [M+Na]+ calcd for C16H15F3N2O2Na 347.0978, found 347.0975.

(R)-2,2,2-trifluoro-N-(1-(1-methyl-1H-pyrrol-2-yl)-1-oxo-3-phenylpropan-2-yl)acetamide

(TFA-D-Phe-(2-(N-Me)Pyr), D-95): Yellowish amorphous mass; [α]D = –53 (c 1.0, CHCl3).

IR (neat) : 3264, 3123, 3088, 1725 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.37 (1H, br s, NH),

7.26–7.20 (4H, m, Ar-H), 7.03–6.99 (2H, m, Ar-H & CH), 6.62 (2H, d, J = 2.0 Hz, CH), 5.33

(1H, dd, J = 13.8, 6.4 Hz, CHNH), 3.67 (3H, s, CH3), 3.31 (2H, dd, J = 13.8, 6.4 Hz, CH2CH),

3.15 (1H, dd, J = 13.8, 6.4 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz, CDCl3) δ: 189.8, 156.4

(q, 2JCF = 37.6 Hz), 135.1, 129.5 (2 x CH), 128.3 (2 x CH), 128.0, 127.1, 124.1, 122.3, 115.7

(q, 1JCF = 287.7 Hz), 109.8, 56.2, 39.2, 36.7 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd for

C16H15F3N2O2Na 347.0978, found 347.0976.

(S)-2,2,2-trifluoro-N-(1-(1-methyl-1H-pyrrol-3-yl)-1-oxo-3-phenylpropan-2-yl)acetamide

(TFA-L-Phe-(3-(N-Me)Pyr), L-96): Yellowish amorphous mass; [α]D = +48 (c 1.0, CHCl3).

IR (neat) : 3307, 1704, 1654 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.25–7.21 (3H, m, Ar-H),

7.03 (1H, d, J = 4.3 Hz, Ar-H), 7.00–6.96 (2H, m, Ar-H & CH), 6.93 (1H, s, CH), 6.20 (1H,

dd, J = 4.3, 2.3 Hz, CH), 5.48 (1H, dd, J = 13.8, 5.6 Hz, CHNH), 3.90 (3H, s, CH3), 3.34 (1H,

dd, J = 13.8, 5.6 Hz, CH2CH), 3.13 (1H, dd, J = 13.8, 5.6 Hz, CH2CH) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 185.0, 156.3 (q, 2JCF = 37.1 Hz), 135.0, 133.0, 129.4 (2 x CH), 128.4 (2 x

CH), 127.8, 127.3, 120.9, 115.7 (q, 1JCF = 287.7 Hz), 109.2, 55.4, 40.0, 37.6 ppm. HRMS-ESI

(m/z) [M+Na]+ calcd for C16H15F3N2O2Na 347.0978, found 347.0979.

(R)-2,2,2-trifluoro-N-(1-(1-methyl-1H-pyrrol-3-yl)-1-oxo-3-phenylpropan-2-yl)acetamide

(TFA-D-Phe-(3-(N-Me)Pyr), D-96): Yellowish amorphous mass; [α]D = –48 (c 1.0, CHCl3).

IR (neat) : 3302, 1703, 1649 cm-1

. 1H-NMR (270 MHz, CDCl3) δ: 7.25–7.22 (3H, m, Ar-H),

7.03 (1H, d, J = 4.0 Hz, Ar-H), 7.00–6.96 (2H, m, Ar-H & CH), 6.93 (1H, s, CH), 6.20 (1H,

dd, J = 4.3, 2.3 Hz, CH), 5.48 (1H, dd, J = 13.8, 5.6 Hz, CHNH), 3.90 (3H, s, CH3), 3.34 (1H,

dd, J = 13.8, 5.6 Hz, CH2CH), 3.13 (1H, dd, J = 13.8, 5.6 Hz, CH2CH) ppm. 13

C-NMR (67.5

MHz, CDCl3) δ: 185.0, 156.3 (q, 2JCF = 37.4 Hz), 135.0, 133.0, 129.4 (2 x CH), 128.4 (2 x

CH), 127.8, 127.3, 120.9, 115.7 (q, 1JCF = 287.7 Hz), 109.2, 55.4, 40.0, 37.6 ppm. HRMS-ESI

(m/z) [M+Na]+ calcd for C16H15F3N2O2Na 347.0978, found 347.0980.

140

(S)-2,2,2-trifluoro-N-(3-(4-hydroxyphenyl)-1-oxo-1-phenylpropan-2-yl)acetamide (TFA-

L-Tyr-Ph, L-100): Colorless amorphous mass. [α]D = +45 (c 1.0, MeOH). IR (neat) : 3464,

3311, 1713, 1682 cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.65 (1H, br d, J = 7.3 Hz,

NH), 8.25 (1H, s, OH), 8.07 (2H, d, J = 7.4 Hz, Ar-H), 7.67 (1H, t, J = 7.4 Hz, Ar-H), 7.56

(2H, t, J = 7.4 Hz, Ar-H), 7.11 (2H, d, J = 8.2 Hz, Ar-H), 6.76 (2H, d, J = 8.2 Hz, Ar-H), 5.77

(1H, ddd, J = 8.9, 7.3, 5.2 Hz, CHNH), 3.26 (1H, dd, J = 14.2, 5.2 Hz, CH2CH), 3.00 (1H, dd,

J = 14.2, 8.9 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz, ACETONE-D6) δ: 197.4, 157.2, 157.2

(q, 2JCF = 36.9 Hz), 135.8, 134.6, 131.2 (2 x CH), 129.7 (2 x CH), 129.3 (2 x CH), 127.8,

116.9 (q, 1JCF = 287.7 Hz), 116.1 (2 x CH), 57.0, 36.9 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd

for C17H14F3NO3Na 360.0818, found 360.0820.

(R)-2,2,2-trifluoro-N-(3-(4-hydroxyphenyl)-1-oxo-1-phenylpropan-2-yl)acetamide (TFA-

D-Tyr-Ph, D-100): Colorless amorphous mass. [α]D = –45 (c 1.0, MeOH). IR (neat) : 3464,

3309, 1710, 1677 cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.64 (1H, br d, J = 7.6 Hz,

NH), 8.24 (1H, s, OH), 8.07 (2H, d, J = 7.4 Hz, Ar-H), 7.67 (1H, t, J = 7.4 Hz, Ar-H), 7.56

(2H, t, J = 7.4 Hz, Ar-H), 7.11 (2H, d, J = 8.6 Hz, Ar-H), 6.76 (2H, d, J = 8.6 Hz, Ar-H), 5.77

(1H, ddd, J = 8.6, 7.6, 5.1 Hz, CHNH), 3.26 (1H, dd, J = 14.3, 5.1 Hz, CH2CH), 3.00 (1H, dd,

J = 14.3, 8.6 Hz, CH2CH) ppm. 13

C-NMR (67.5 MHz, ACETONE-D6) δ: 197.4, 157.2, 157.2

(q, 2JCF = 37.4 Hz), 135.8, 134.6, 131.2 (2 x CH), 129.7 (2 x CH), 129.4 (2 x CH), 127.8,

116.9 (q, 1JCF = 287.2 Hz), 116.1 (2 x CH), 57.0, 36.9 ppm. HRMS-ESI (m/z) [M+Na]

+ calcd

for C17H14F3NO3Na 360.0818, found 360.0814.

2,2,2-trifluoro-N-(2-(4-hydroxyphenyl)-1-phenylethyl)acetamide (101): Yellowish

amorphous mass. IR (neat) : 3329, 1693 cm-1

. 1H-NMR (270 MHz, ACETONE-D6) δ: 8.82

(1H, br d, J = 8.6 Hz, NH), 8.18 (1H, s, OH), 7.44 (2H, d, J = 7.9 Hz, Ar-H), 7.38–7.24 (3H,

m, Ar-H), 7.09 (2H, d, J = 8.6 Hz, Ar-H), 6.74 (2H, d, J = 8.6 Hz, Ar-H), 5.24 (1H, ddd, J =

8.6, 8.6, 6.9 Hz, CHNH), 3.15 (1H, d, J = 8.6 Hz, CH2CH), 3.12 (1H, d, J = 6.9 Hz, CH2CH)

ppm. 13

C-NMR (67.5 MHz, ACETONE-D6) δ: 156.9, 156.8 (q, 2JCF = 36.3 Hz), 142.3, 131.0

(2 x CH), 129.4 (2 x CH), 129.3 (2 x CH), 128.3, 127.7, 117.1 (q, 1JCF = 288.3 Hz), 115.9 (2 x

CH), 56.8, 41.5 ppm.

141

3.4 Conclusion

To summarize, through the reaction of aliphatic TFA-protected -amino acid-OSu (L-

/D-33, L-/D-38, 53, L-/D-57, L-/D-61, L-/D-65, L-/D-69, L-/D-73, and L-/D-77) with arenes

under conventional Friedel–Crafts conditions, it is more convenient to synthesize chiral TFA-

protected -amino aryl-ketones as its skeleton can be obtained from optically active material.

The TFA-protected -amino acid-OSu showed high reactivity and is easier to handle during

the reaction than acid chloride. Utilization of TFA-α-amino acid-OSu derivatives having a

side chain of an aromatic moiety (L-/D-82 and L-/D-99) for acyl acceptor in Friedel–Crafts

acylation reaction with AlCl3 as a catalyst might introduce an inter-molecular reaction with

arenes and heterocycles. Rapid intra-molecular cyclization is possible in the reaction for TFA-

L-/D-Phe-OSu (L-/D-82). All TFA-protected -amino acid-OSu can contribute as an excellent

acyl donor to Friedel–Crafts acylation without loss of chirality at the α-carbon. This typical

reaction can play an important role in further study to broaden application for the synthesis of

new bioactive compounds. The novel compounds explored in this study also can contribute to

new synthetic pathways and can be a part of the comprehensive future study to their

exploitation in the organic synthesis, for example studying the complex bio-synthesis of

acetyl-CoA in human from simple -amino aryl ketone starting material.

142

Chapter 4 Hydrogen/deuterium exchange of aromatic compounds

(cycloDOPA derivatives)

4.1 Introduction

CycloDOPA (5,6-dihydroxy-indoline-2-carboxylic acid, leukodopachrome, cDOPA,

102, Scheme 32), known as metabolites of aromatic α-amino acid of tyrosine,111

is one of

intermediate in eumelanin pathway (melanin formation in mammalian)112–114

and main

skeleton of betanidin (betalain pigment in plant).115,116

In spite of its simple structure, which

can be constructed from intra-molecular cyclization of L-DOPA 103, a few studies have been

reported for the synthesis and its commercial sources are also limited. First synthesis of

cDOPA has been reported in 1968.117

Based on this study, the skeleton of cDOPA was

generated once at early stage and isolated as its peracetylated form, O,O,N-triacetyl cDOPA

methyl ester (Ac3-cDOPA-OMe, 104, Scheme 32).118

Scheme 32 Synthesis of cDOPA 102

Hydrogen/deuterium (H/D) exchange, the displacement of hydrogen bonded to carbon

by deuterium, is one of the methods to broaden the variety of isotopically labeled material.119

Based on the recent progress of the mass equipment, H/D exchange also provides analysis

with stable isotopes to reveal the biosynthetic pathways.120

Previously, the introduction of

deuterated trifluoroacetic acid (TFA-d) for H/D exchange of hydrogen in the aromatic ring of

cDOPA was reported.117

The selective H/D exchange (within ~50% deuterium incorporation)

of hydrogen in the aromatic moiety can be formed only at 7-position after the treatment at 83

oC, but no incorporation of deuterium was found when treated at 70

oC.

143

Although the deuterium incorporation in cDOPA can be utilized for analysis of me

metabolic pathway, to date, the synthesis of fully deuterated cDOPA had not been studied. An

acid-catalyzed H/D exchange is known as an efficient method for incorporating deuterium

into an aromatic moiety.43,119

Recently, our detail analysis of chemical stability for cDOPA

revealed that cDOPA was stable in acidic conditions and easily broken over pH 4 to be

converted dihydroxyindole derivatives.118

Moreover, deprotection of Ac3-cDOPA-OMe 104

under acidic condition enabled formation of cDOPA 102 also to demonstrate stability of

cDOPA 118

that indicates its potential for H/D exchange in acidic condition. Since HCl system

can be applied for deacetylation to result in cDOPA (Scheme 32), deuterium chloride (DCl)

system is expected to introduce deuterium to cDOPA moiety.

Triflic acid (TfOH), or so-called superacid, is known for its ability as a catalyst that acts

as solvent for utmost amino acids.34

The recent report found that deuterated triflic acid

(TfOD) can be used for H/D exchange of aromatic amino acids such as phenylalanine,

tyrosine,49

or L-DOPA50

under mild conditions to give over 90% deuterium incorporation

only in its aromatic moiety. Since cDOPA is stable at acidic condition, TfOD also can be

introduced for synthesis of deuterium-labeled cDOPA derivatives. Here in this study, the

comprehensive H/D exchange and analysis of deuterium incorporation for cDOPA is studied.

The stable and readily synthesized cDOPA can be utilized for synthesis of the novel fully-

deuterated aromatic moiety of cDOPA derivatives via H/D exchange. The introduction of DCl

and TfOD system is expected to broaden the formation study of deuterium-labeled cDOPA

derivatives.

4.2 Results and Discussion

The cDOPA 102 can be synthesized from oxidation of commercially available L-DOPA

103 and isolated as its peracetylated form of O,O,N-triacetyl cDOPA methyl ester (Ac3-

cDOPA-OMe, 104, Scheme 32).117

The further deacetylation of Ac3-cDOPA-OMe 104 under

strongly acidic conditions at 80 oC represents the stability of cDOPA 102

118 is feasible for

H/D exchange. Since acid-induced H/D exchange,46

such as utilization with deuterium

chloride (DCl) or deuterated triflic acid (TfOD), is known as the convenient method for H/D

exchange of aromatic moiety, two possible exchangeable protons in aromatic ring of cDOPA

are enable to undergo H/D exchange at 4- and 7-positions. The skeleton of cDOPA 102 was

priorly subjected to 2D NMR analysis for ascertaining its each counterpart. Based on 1H-

NMR, the aromatic proton at 4-position was shown as a singlet at δH 6.84 ppm, while proton

at 7-position was shown as a singlet at δH 6.90 ppm, both of which were distinguished by

HMBC analysis (Figure 29).

144

Figure 29 Selected HMBC (500 MHz, D2O) of cDOPA 102

Since the deacetylation of Ac3-cDOPA-OMe 104 by 6 M HCl was conducted at 80 oC

allowing the formation of cDOPA 102 with a fine yield (Scheme 32), the H/D exchange for

synthesis of deuterium-labeled cDOPA derivatives was attempted by using DCl system.

Accordingly, Ac3-cDOPA-OMe 104 was first subjected to 20% DCl/D2O at 80 oC and the

deuterium incorporated form of its unprotected cDOPA 102 was observed by 1H-NMR. In

Figure 30 (b), when 20% DCl/D2O was utilized, the aromatic hydrogen of cDOPA at the 7-

position can be fully deuterated (99% D) meanwhile the 4-position was deuterated up to 60%

by 4 h. In contrast, deuteration of the 4-position was hampered meanwhile 7-position almost

fully deuterated within less than 1 h. These large differences in the deuterium incorporation

between 4- and 7-positions cannot be maintained due to long H/D exchange time to increase

deuterium incorporation at the 4-position (Figure 30 (b)).

Utilization of 5% and 10% DCl/D2O as the lower DCl concentration showed the 7-

position was deuterated almost up to twice higher than the 4-position dependent to time

H-4/C-2

H-4/C-3

H-7/C-8

H-4/C-5

H-7/C-6

145

(Figure 30 (c) and (d)). In contrast to 20% DCl/D2O utilization, lower DCl concentration

needed longer reaction time for H/D exchange at the 7-position. At the 7-position of cDOPA

derivative, deuterium exchange takes place approximately 80% and 95% after 6 h when 5%

and 10% DCl/D2O were utilized, respectively. Thus, 5% or 10% DCl/D2O is considered to be

less suitable for H/D exchange of cDOPA.

Figure 30 Deprotection and hydrogen/deuterium exchange of O,O,N-triacetyl cDOPA methyl

ester (104) at 80 oC dependent on concentration of (a) 35% DCl/D2O, (b) 20% DCl/D2O, (c)

5% DCl/D2O, and (d) 10% DCl/D2O. The time-course of H/D exchange for cDOPA (102)

was determined by 1H NMR.

When 35% DCl/D2O was used at 80 oC (Figure 30 (a)), hydrogen of aromatic ring at the

4- and the 7-position were fully deuterated (99% deuterium incorporation) within 4 h. The

high concentration of DCl is effective for rapid H/D exchange of both aromatic hydrogen

(a) 35% DCl/D2O (b) 20% DCl/ D2O

(c) 5% DCl/D2O (d) 10% DCl/D2O

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D I

nco

rpo

rati

on

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

146

atoms of cDOPA at the 4- and 7-positions which are suitable for synthesizing the fully-

deuterated aromatic ring of cDOPA derivative. Thus, 35% DCl/D2O was subjected to Ac3-

cDOPA-OMe 104 for synthesis of the fully deuterated cDOPA (105, Scheme 33). Compound

105 can be isolated by evaporation and removal of excess DCl with CH3CN washing. During

the isolation process, no loss of deuterated counterpart was observed by 1H-NMR. Compound

105 is stable and feasible to undergo 13

C-NMR measurement in D2O aqueous solution.

Scheme 33 Synthesis of deuterated cDOPA derivative (105)

Deuterium incorporation (% D) for each position is shown in square brackets.

For further understanding of selective H/D exchange on cDOPA, unprotected cDOPA

102 was also directly subjected to H/D exchange at various temperatures. The H/D exchange

of cDOPA 102 was started with utilization of 20% DCl/D2O. This DCl concentration is

considered to be a suitable condition for the selective deuteration of cDOPA, since within less

than 1 h of Ac3-cDOPA-OMe 104 subjection, the differences of H/D exchange at 4- and 7-

positions can be observed (Figure 30 (a)). When cDOPA 102 was subjected to the reaction

20% DCl/D2O at 80 oC, hydrogen at the 7-position of aromatic cDOPA can be fully

deuterated meanwhile the 4-position can be only deuterated approximately 40% after 4 h. The

slower H/D exchange within the same DCl concentration and temperature for direct cDOPA

102 (Figure 31 (a)) rather than subjection of Ac3-cDOPA-OMe 104 (Figure 30 (a)) might be

due to the presence of accumulated acetic acid as byproduct of deacetylation that increases the

system of acidity during H/D exchange to enhance condition for the H/D exchange.

When the 7-position can be fully deuterated after 4 h, decrease of temperature to 70 oC

resulted in deuteration of cDOPA aromatic moiety at the 4-position as only 30% or so (Figure

31 (b)). Similarly, the 4-position of cDOPA showed approxymately 20% deuterium

incorporation while the 7-position showed nearly 90% deuterium incorporation after 6 h when

H/D exchange was conducted at 60 oC (Figure 31 (c)). As for 50

oC (Figure 31 (d)), the same

tendency also was shown within prolonged time, in which the 4-position of cDOPA showed

only around 20% deuterium incorporation and the 7-position showed almost 90% deuterium

incorporation after 12 h of the treatment. These results indicated that by lowering the

temperature, the H/D exchange at the 4-position is slower than the 7-position, which is

147

depending on H/D exchange time. The further subjection of cDOPA 102 with 20% DCl at 60

oC for 6 h, followed by evaporation and removal of excess HCl, can result in deuterated

cDOPA derivative at the 7-position (compound 106, Scheme 34). The selective deuterium-

labeled of compound 106 is stable and deuterated counterparts at 7-position (88% D) and 4-

position (23% D) also showed no loss of deuterium incorporation during the isolation process.

Figure 31 Hydrogen/deuterium exchange of cDOPA (102) by 20% DCl/D2O dependent on

temperature of (a) 80 oC, (b) 70

oC, (c) 60

oC, and (d) 50

oC.

(a) 80 oC (b) 70 oC

(c) 60 oC (d) 50 oC

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12

D I

nco

rpora

tion

(%

)

Time (h)

H-4 H-7

148

Scheme 34 Synthesis of deuterated cDOPA derivative (106)

a Recovery percentage. Deuterium incorporation (% D) for each position is shown in

square brackets.

Triflic acid (TfOH) is categorized as a strong Brønsted acid and known for its ability to

solve mostly amino acid skeleton.34,49,121

The deuterated triflic acid (TfOD) can be used to

promote H/D exchange34

of aromatic amino acids, such as phenylalanine, tyrosine,49

or L-

DOPA50

under a mild condition (rt or 0 oC) to give over 90% deuterium incorporation only in

its aromatic moiety. To complete the synthesis of deuterated cDOPA derivatives, cDOPA 102

was then treated with TfOD. At room temperature (Figure 32 (a)), approximately 90%

deuterium incorporation takes place at the 4- and 7-positions of cDOPA aromatic ring after 1

h. The degree of exchange did not change even with longer incubation time.

Figure 32 Hydrogen/deuterium exchange of cDOPA (102) with triflic acid (TfOD, 40 equiv.)

at (a) rt and (b) 0 oC

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6

D in

corp

ora

tio

n(%

)

Time (h)

H-4 H-7

(a) rt (b) 0 oC

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7

D in

corp

ora

tion

(%)

Time (h)

H-7 H-4

149

In Figure 32 (b), H/D exchange of cDOPA at lower temperature (0 oC) showed faster

H/D exchange at the 4-position than the 7-position. Within 40 min, proton at the 4-position

deuterium can be achieved as 84% D and the 7-position was deuterated 36% D based on

observation with 1H-NMR. Elongation time for H/D exchange at 0

oC showed that the two

sites can be deuterated approximately 90% after 6 h. TfOD is suitable for H/D exchange of

cDOPA aromatic moiety at room temperature (nearly 90% deuterium incorporation after 1 h,

Figure 32 (a)), compared with previous utilization of TFA-d that needed high temperature to

conduct only 50% deuterium at the 7-position.117

It is possible due to TfOD known to be

relatively strong acid rather than TFA-d.34,122

By utilizing TfOD at room temperature for 1 h,

full deuterated aromatic moiety of cDOPA derivatives 107 can be formed (Scheme 35 (a)).

Next, the selective deuterated cDOPA at the 4-position of compound 108 can be synthesized

by utilizing TfOD at 0 oC for 40 m (Scheme 35 (b)). The treatments of pH adjustment with

NaOD, freeze drying, and then gel filtration chromatography (Sephadex G-10 eluted with HCl

(pH = 3)) were conducted for the isolation of deuterated cDOPA (107 and 108) derivatives

from TfOD utilization (See Figure 33 for details). The attempt to remove of excess TfOD was

difficult to conduct after all further treatments, although deuterium incorporation of

deuterated cDOPA derivatives using TfOD showed no significant differences in the deuterium

incorporation (% D) during the process.

Scheme 35 Synthesis of deuterated cDOPA derivatives (107 and 108)

a %Yield measured from UV-Vis spectra (TfOD/D2O). Deuterium incorporation (% D) for

each position is shown in square brackets.

150

Index:

C1: cDOPA 102 is treated with TfOD at 0 oC for 40 m and directly subjected into NMR.

P1: C2 after pH (2–3) adjustment by NaOD.

F1: P2 after overnight freeze drying.

G1: F1 after separation by gel filtration chromatography (Sephadex G-10 eluted by HCl (pH = 3), 27% recovery

in Cl- salt measured from UV-Vis spectra).

C2: cDOPA 102 is treated with TfOD at rt for 1 h and directly subjected into NMR.

P2: C2 after pH (2–3) adjustment by NaOD.

F2: P2 after overnight freeze drying.

G2: F2 after separation by gel filtration chromatography (Sephadex G-10 eluted by HCl (pH = 3), 26% recovery

in Cl- salt measured from UV-Vis spectra).

Figure 33 Excess TfOD removal trials for deuterated cDOPA derivatives (107 and 108)

0

10

20

30

40

50

60

70

80

90

100

D In

corp

ora

tio

n (

%)

4-position 7-position

0 oC, 40 m rt, 1 h

C1 P1 F1 G1 C2 P2 F2 G2

pH (2-3) adjustment – ✓ ✓ ✓ – ✓ ✓ ✓

Freeze drying – – ✓ ✓ – – ✓ ✓

Gel filtration – – – ✓ – – – ✓

151

The fully and partially deuterated cDOPA derivatives (105–108) via H/D exchange can

be formed by the utilization of DCl or TfOD system. The deuterium counterpart of deuterated

cDOPA derivatives of 105–108 can be determined by 1H-NMR (Figure 34). Based on

1H-

NMR (Figure 34), there is no change of proton NMR signal integrations corresponding to the

heterocyclic side chain of deuterated cDOPA derivatives. Thus, the H/D exchanges indicated

occurred only specific in aromatic moiety of cDOPA. H/D exchange is basically complex

process with complicated intermediate that can be affected by temperature, deuterium source,

and pH. Thus, the site-selectivity between H/D exchanges of the 4- and 7-positions on the

aromatic ring of cDOPA treated with DCl or TfOD possibly due to differences of acidity. H-4

is suggested to be more difficult to conduct H/D exchange than H-7 and since TfOH is

categorized as superacid which has higher acidity than HCl, thus at lower temperature H-4

can be selectively deuterated. Moreover, H/D exchange in TfOD system might occur

selectively due to intra-molecular exchange between two sites that have approximately equal

proton affinity in their conjugate acid form (1,2-hydride shifts where the hydrate is transferred

in the ring plane).123

However, the controllable deuterated cDOPA derivatives can be used to

infer biological macromolecules due to its beneficial detection with spectroscopic methods.

Figure 34 Selected 1H-NMR profile of cDOPA (102) and deuterated cDOPA derivatives

(105–106, 108)

7 4

1

4

5

7

(DCl 35%, 80 oC, 4 h)

(DCl 20%, 60 oC , 6 h)

(TfOD, 0 oC, 40 m)

ppm

7 4

(DCl 35%, 80 oC, 4 h)

(DCl 20%, 60 oC, 6 h)

(TfOD, 0 oC, 40 m)

102

105

106

108

152

4.3 Experimental section

General methods. 1H-NMR spectra were measured by a Jeol EX 270 spectrometer

(JEOL, Tokyo, Japan) for determining the H/D exchange proportion. MS data were obtained

with a Waters LCT Premier XE instrument. HRMS-ESI spectra were obtained with a Waters

UPLC ESI-TOF mass spectrometer (Waters, Milford, CT, USA). All reagents used were of

analytical grade. Optical rotations were measured at 23 oC on JASCO DIP370 polarimeter

(JASCO, Tokyo, Japan). TfOD (98% D) was purchased from Sigma–Aldrich. DCl (35 wt% in

D2O, 99% D) was purchased from Wako. The cDOPA derivatives (102 and 104) were

synthesized by reported methods with slight modification.

L-DOPA methyl ester hydrochloride. The SOCl2 (5.0 mL, 70 mmol) was added

slowly at –5 oC dry MeOH (20 ml). L-DOPA (103, 1.00 g, 50 mmol) was added to the

reaction mixture. The reaction mixture was stirred at rt for 1 h and refluxed at 85 oC for 1 h.

After the reaction, the reaction mixture was concentrated to afford colorless amorphous mass

of L-DOPA methyl ester hydrochloride that was directly used for the next step without further

purification.

O,O,N-Triacetyl cDOPA methyl ester (104). To a chilled solution of L-DOPA methyl

ester hydrochloride (800 mg, 3.230 mmol) in 88 mM phosphate buffer (400 ml; pH 8,

prepared by mixing 0.4 g of KH2PO4 and 9.57 g of Na2HPO4 in 800 ml water), a solution of

K3[Fe(CN)6] (6.48 g, 19.68 mmol) in phosphate buffer (200 ml) was added at 4 °C. After 8

sec, a solution of Na2S2O4 (5.12 g, 29.41 mmol) in phosphate buffer (100 ml) at 4 °C was

added and stirred for 30 sec. The reaction mixture was made pH 1 with concentrated HCl,

concentrated and co-evaporated with toluene for several times. The residue was suspended in

Ac2O (40 ml) and pyridine (40 ml) and the suspension was stirred at room temperature for 4 h,

then filtered by Celite®. The insoluble material was washed with CH2Cl2 and the filtrate was

concentrated. The residue was partitioned between CH2Cl2 (80 ml) and 1 M HCl (80 ml). The

organic layer was washed with sat. NaHCO3, water and brine, dried over MgSO4, filtrated,

and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2 /

MeOH = 80 / 1) to afford O,O,N-triacetyl cDOPA methyl ester (104): Colorless amorphous

mass. [α]D = –73 (c 1, CHCl3), 1H-NMR (270 MHz, CDCl3) δ: 8.06 (1H, s), 6.99–6.95 (1H,

m), 5.18 (0.3 H, d, J = 8.6 Hz), 4.94 (1H, d, J = 8.6 Hz), 3.78 (3H, s), 3.57 (1H, dd, J = 17.0,

10.7 Hz), 3.25 (1H, d, J = 16.5 Hz), 2.29– 2.11 (9H, m) ppm. 13

C-NMR (67.5 MHz, CDCl3)

δ: 171.0 , 168.6 , 168.0, 167.8 , 140.7 , 140.2 , 137.7 , 126.5 , 118.5 , 111.7 , 61.2 , 52.5 , 32.4 ,

22.9 , 20.0, 20.0 ppm. HRMS (ESI): calcd for C16H18NO7 [M + H]+ 336.1083, found

336.1086.

153

cDOPA hydrochloride (102, cDOPA). O,O,N-Triacetyl cDOPA methyl ester (104,

134.5 mg, 0.40 mmol) was dissolved in 20% HCl. The reaction mixture was stirred at 80℃

for 4 h, and concentrated. The residue was washed with CH3CN to remove excess HCl to

afford product as brown amorphous mass (88.0 mg, 0.38 mmol, 95%). [α]D = –85 (c 1.0,

H2O), ref.118

[α]D = –91.4 (c 0.5 H2O). 1H-NMR (270 MHz, D2O) δ: 6.90 (1H, s), 6.84 (1H, s),

4.85 (1H, dd, J = 9.7, 6.8 Hz), 3.50 (1H, dd, J = 16.2, 9.6 Hz), 3.27 (1H, dd, J = 16.2, 6.6 Hz)

ppm. 13

C-NMR (67.5 MHz, D2O) δ: 171.6, 146.6, 145.0, 126.5, 125.6, 112.6, 106.9, 61.1,

33.0 ppm. HRMS (ESI): calcd for C9H10NO4 [M + H]+ 196.0610, found 196.0612.

General procedures for H/D exchange with DCl. The O,O,N-triacetyl cDOPA methyl

ester (104, 45 mg, 0.13 mmol) or cDOPA (102, 45 mg, 0.19 mmol) was dissolved in DCl

(2.34 mL) and stirred at specific temperature (mentioned in Figure 30 and 31). The part of

reaction mixture (250 μL) was diluted with D2O (200 μL) in ice bath and then subjected to

1H-NMR, depending on time.

General procedures for H/D exchange with TfOD. The cDOPA (102, 45 mg, 0.19

mmol) was dissolved in TfOD (672 μL, 7.6 mmol) and stirred at specific temperature

(mentioned in Figure 32). The part of reaction mixture (67.2 μL) was diluted with D2O (500

μL) for subjection to 1H-NMR, depending on time.

cDOPA-4,7-d2 (105) The O,O,N-triacetyl cDOPA methyl ester (104, 49.3 mg, 0.15

mmol) was dissolved in 35% DCl (2.56 mL, 28.7 mmol). The reaction mixture was stirred at

80 oC for 4 h, and concentrated under low pressure. The residue was washed with MeCN and

diluted with a small amount of D2O, and then centrifuged. The supernatant liquid then

concentrated to afford product as brown amorphous mass (32.1 mg, 0.14 mmol, 94%). [α]D –

73 (c 1.0, D2O). 1H-NMR (270 MHz, D2O) δ: 6.90 (0.01H, s), 6.82 (0.01H, s), 4.89 (1H, t, J =

9.7 Hz), 3.50 (1H, dd, J = 16.2, 9.6 Hz), 3.28 (1H, dd, J = 16.2, 6.6 Hz) ppm. 13

C-NMR (67.5

MHz, D2O) δ: 172.4, 146.8, 145.1, 127.0, 126.0, 112.6 (J = 21.2 Hz), 106.9 (J = 23.2 Hz),

61.5, 33.3 ppm. HRMS (ESI): calcd for C9H8D2NO4 [M + H]+ 198.0735, found 198.0724.

cDOPA-7-d (106) The cDOPA (102, 49.2 mg, 0.21 mmol) was dissolved in 20% DCl

(2.56 mL, 16.6 mmol). The reaction mixture was stirred at 60 o

C for 6 h, and concentrated

under low pressure. The residue was washed with MeCN and diluted with a small amount of

D2O, then centrifuged. The supernatant liquid was then concentrated to afford product as

brown amorphous mass (48.2 mg, 0.21 mmol, 98%). [α]D –76 (c 1.0, D2O). 1H-NMR (270

MHz, D2O) δ: 6.90 (0.12H, s), 6.80 (0.77H, s), 4.91 (1H, t, J = 7.9 Hz), 3.50 (1H, dd, J = 16.2,

9.6 Hz), 3.27 (1H, dd, J = 16.2, 6.6 Hz) ppm. 13

C-NMR (67.5 MHz, D2O) δ: 172.3, 146.8,

145.1, 126.8, 126.0, 112.8, 106.7 (J = 23.2 Hz), 61.5, 33.3 ppm. HRMS (ESI): calcd for

C9H9DNO4 [M + H]+ 197.0673, found 197.0666.

154

cDOPA-4-d (108) The cDOPA (102, 4.4 mg, 0.019 mmol) was dissolved in TfOD (67.2

μL, 0.76 mmol) and stirred at 0 oC for 40 min. The reaction mixture was diluted with D2O

(500 μL), measured the deuterium incorporation by 1H-NMR and then UV-Vis spectroscopy

(UV (TfOD/D2O): λmax (ε) = 285 (4328) nm, 99%). [α]D –78 (c 0.78, TfOD/D2O). 1H-NMR

(270 MHz, D2O) δ: 6.90 (0.64H, s), 6.84 (0.16H, s), 4.95 (1H, dd, J = 9.7, 6.8 Hz), 3.53 (1H,

dd, J = 16.2, 9.6 Hz), 3.32 (1H, dd, J = 16.2, 6.8 Hz) ppm. 13

C-NMR (67.5 MHz, D2O) δ:

173.1, 146.8, 145.2, 127.2, 126.3, 112.8 (J = 21.2 Hz), 107.3, 62.0, 33.5 ppm. HRMS (ESI):

calcd for C9H9DNO4 [M + H]+ 197.0673, found 197.0679.

4.4 Conclusion

In conclusion, H/D exchange of cDOPA derivatives under DCl and TfOD allowing the

formation of fully or partially deuterated cDOPA derivatives (105–108). When DCl was

utilized at 60 oC, H/D exchange of cDOPA shifted at the 7-position was faster than 4-position.

In contrast, utilization of TfOD at 0 oC enabled the H/D exchange at the 4-position to be faster

than the 7-position of aromatic cDOPA moiety. Isolation of the deuterated cDOPA derivatives

formed by the H/D exchange reaction utilizing DCl was simpler than those obtained by TfOD

subjection. H/D exchange in cDOPA might simplify the analysis of complex biomolecules

with stable isotopes to reveal the biosynthetic pathways of cDOPA, for example in the

biosynthesis of betanin in plants or melanine in mammals.

155

Chapter 5 Conclusion and future prospect

Natural products offer a privileged starting point in the search for highly specific and

potent modulators of biomolecular function. Optimization of organic synthesis has the power

to utilize chemical features of natural products in the laboratory and applies their developed

synthetic strategies and technologies to construct novel compounds. Comprehensive study for

exploration of structurally diverse variance of carbohydrates and -amino acids in this study

can impact the chemical biology and drug development.

Direct halogenation of unprotected carbohydrates, such as sucrose and 1-kestose,

employed the selective reaction at their primary alcohols only. By modifying the reagents for

this typical reaction, the reactivity can be controlled, which can lead to completion of the

structure analysis, revision of the previously elucidated ambiguous structure, and also

exploration for synthesis of new compounds. It is an endeavoring study to distinguish the

positions of the halogenations for unprotected carbohydrate by mass spectrometry or NMR

analyses. Especially, the first synthesis of selectve halogenation for sucrose has been reported

three decades ago. The NMR analysis of the mono-halogenated products has not been fully

assigned and none of the study can doubt the reported results until now. The careful

purification and identification for protected carbohydrates are important; therefore utilization

of another protecting group, such as methyl or benzyl ether, in the next study should also be

taken for consideration. Since position of the specific halogenations can influence certain

sweetness acivity, it also suggests that the introduction of halogenated sucrose and 1-kestose

at their primary positions is potentially tested their sweetness activity and might be use as

alternative sweetener for diabetics.

Introduction of representative acyl donors for Friedel–Crafts acylation which conducts

the C-terminal activation of N-protected -amino acids, with TFA--amino N-

hydroxysuccinimide ester, offered convinient methods for synthesis of TFA--amino-aryl

ketone. The extensive acylation by this functional group administered the chance for

expansion of new structural design that is suitable for formation of acylium cation, and its

stability can be considered as more efficient method than the use of acid chlorides. Several

similar active esters, such as pentafluorophenyl diphenylphosphinate (FDPP), are potentially

to be used as acylation reagents due to its reactivity. Moreover, demonstration of isoleucine

and its diastereomer for checking the chirality center during the modification held a

convinient chemical feature in peptide synthesis in future study. The resulted TFA--amino-

156

aryl ketone also can contribute to prelimiary study of drug design or biomolecular

identification.

H/D exchange on aromatic moiety is far more advance in reaserch for so-called

deuterated medicine synthesis. The success of selective deuteration on aromatic moiety of

cDOPA derivatives can open the perception of H/D exchange condition. Acid-catalyst

utilization using DCl or TfOD is advantageous for homogenous H/D exchange reaction. The

new finding of another homogenous H/D exchange catalyst suggested that rare metal super

acids, such as lanthanide triflates should be explored. Since TfOD separation from reaction

mixture is complicated for cDOPA derivatives due to its stablity in acidic solvent,

heterogenous reaction system is highly recommeded in future study. The use of TfOH

mediated SiO2 had been explored and might be useful for heterogenous H/D exchange if

deuterium can be introduced in the system. Moreover, deuterated aromatic moiety of cDOPA

derivatives can be utilized for revealing the mechanism of natural product bio-synthetic

pathways in plant or mamalian.

Since synthesis of α-amino-aryl ketone via Friedel–Crafts acylation required activation

by acid catalyst and synthesis of deuterium-labelled compounds usually conducted by the use

of acid as deuterium source, preparation of selective deuterium incorporated at aromatic

moiety of α-amino-aryl ketone is suggested. Introduction for one-step acylation and H/D

exchange reaction is recommended by utilizing deuterated super acid as catalyst and

deuterium source. Moreover, introduction of deuterium in amino acid derivatives can be

useful in proteomics for expression, identification, and quantification of protein in plants or

animals. The deuterated analogues can also mimic certain enzyme substrate that might be

used as potential approach for investigating enzyme-substrate binding process and revealing

the bioactivity for certain bio-molecule in human.

Direct halogenation of carbohydrate, extensive acylation of -amino acids, and selective

H/D exchange of aromatic compounds are taken into comprehensive studies of organic

synthesis that was utilized chemical features of natural products. These establishements are

beneficial in the field of molecular chemistry and allowing for modification of vast structure

deversity for major biomolecules, including sugars an protein. Thus, the collaboration study

for multipurpose laboratory researches and commercial needs can support the development of

organic synthesis and modification of natural products based on its chemical features, and it

should be considered for economic benefits.

157

Reference

(1) Brown, W. H.; Foote, C. S.; Iverson, B. L.; Anslyn, E. V. Organic chemistry, Sixth

Edition; Brooks/Cole, Cengage Learning: Brooks/Cole, USA, 2012.

(2) Queneau, Y.; Fitremann, J.; Trombotto, S. The chemistry of unprotected sucrose: The

selectivity issue. Comptes Rendus Chim. 2004, 7, 177–188.

(3) Hough, L.; Khan, R. Intensification of sweetness. Trends Biochem. Sci. 1978, 3, 61–

63.

(4) Hough, L.; Mufti, K. S. Sucrochemistry Part VI. Further reactions of 6,6′-di-O-

tosylsucrose and a comparison of the reactivity at the 6 and 6′-positions. Carbohydr.

Res. 1972, 25, 497–503.

(5) Khan, R.; Jenner, M. R.; Mufti, K. S. Reaction of methanesulphonyl chloride-N,N-

dimethylformamide with partially esterified derivatives of sucrose. Carbohydr. Res.

1975, 39, 253–262.

(6) Kakinuma, H.; Yuasa, H.; Hashimoto, H. Synthesis of l′,6′-disubstituted sucroses and

their behavior as glucosyl donors for a microbial -glucosyltransferase. Carbohydr.

Res. 1996, 284, 61–72.

(7) Sachinvala, N. D.; Niemczura, W. P.; Litt, M. H. Monomers from sucrose. Carbohydr.

Res. 1991, 218, 237–245.

(8) Gus, R. D. G.; Guthrie, R. D.; Jenkins, I. D.; Watters, J. J. 1′-Derivatives of sucrose

and their acid hydrolysis. Autr. J. Chem. 1980, 33, 2487–2497.

(9) Andrade, M. M.; Barros, M. T.; Rodrigues, P.; Barros, T.; Rodrigues, P.; Barros, M.

T.; Rodrigues, P. Selective synthesis under microwave irradiation of carbohydrate

derivatives containing unsaturated systems. Eur. J. Org. Chem. 2007, 3655–3668.

(10) Barros, M. T.; Petrova, K. T.; Correia-da-Silva, P.; Potewar, T. M. Library of mild and

economic protocols for the selective derivatization of sucrose under microwave

irradiation. Green Chem. 2011, 13, 1897–1906.

(11) Ballard, J. M.; Hough, L.; Richardson, A. C.; Fairclough, P. H. Reaction of sucrose

with sulphuryl chloride. J. Chem. Soc., Chem. Commun. 1972, 1524–1528.

(12) Karl, H.; Lee, C.-K.; Khan, R. Synthesis and reactions of tert-butyldiphenylsilyl ethers

of sucrose. Carbohydr. Res. 1982, 101, 31–38.

(13) Khan, R.; Lal Bhardwaj, C.; Mufti, K. S.; Jenner, M. R. Synthesis of 6,6′-dideoxy-

6,6′-dihalosucroses and conversion of 6,6′-dichloro-6,6′-dideoxysucrose hexa-acetate

into 6,6′-diamino-6,6′-dideoxysucrose. Carbohydr. Res. 1980, 78, 185–189.

(14) Castro, B.; Chapleur, Y.; Gross, B. Alkyloxyphosphonium salts. VII. Selective

activation of 1′-α,α-trehalose and saccharose. Carbohydr. Res. 1974, 36, 412–419.

(15) Anisuzzaman, K. A. M.; Whistler, R. L. Selective replacement of primary hydroxyl

groups in carbohydrates: preparation of some carbohydrate derivatives containing

halomethyl groups. Carbohydr. Res. 1978, 61, 511–518.

(16) Khan, R. Chemistry and new uses of sucrose: How important? Pure Appl. Chem. 1984,

56, 833–844.

(17) Khan, R. Sucrochemistry Part VII. Preparation and reactions of penta-O-

benzoylsucrose 1′,6,6′-tris(chlorosulphate) and hexa-O-benzoylsucrose 6,6′-

bis(chlorosulphate). Carbohydr. Res. 1972, 25, 504–510.

(18) Bolton, C. H.; Hough, L.; R., K. Sucrochemistry Part I. New derivatives of sucrose

prepared from the 6,6′-di-O-tosyl and the octa-O-mesyl derivatives. Carbohydr. Res.

1972, 21, 133–143.

(19) Hough, L.; Mufti, K. S. Sucrochemistry Part IX. Mono-, di-, tri-, and tetra-substituted

derivatives prepared from sucrose octamethanesulphonate. Carbohydr. Res. 1973, 27,

47–54.

158

(20) Hough, L.; Richardson, A. C.; Salam, M. A. The reaction of raffinose with sulphuryl

chloride. Carbohydr. Res. 1979, 71, 85–93.

(21) Linghua, C.; Hui, D.; Guanzhong, S.; Yuting, L. Synthesis of chloro-deoxy-melezitose.

Nat. Prod. Res. Dev. 1999, 11, 10–13 (in Chinese).

(22) Shibata, R.; Kimura, M.; Takahashi, H.; Mikami, K.; Aiba, Y.; Takeda, H.; Koga, Y.

Clinical effects of kestose, a prebiotic oligosaccharide, on the treatment of atopic

dermatitis in infants. Clin. Exp. Allergy 2009, 39, 1397–1403.

(23) Vega, R.; Zúniga-Hansen, M. E. Enzymatic synthesis of fructooligosaccharides with

high 1-kestose concentrations using response surface methodology. Bioresour.

Technol. 2011, 102, 10180–10186.

(24) Yun, J. W. Fructooligosaccharides—Occurrence, preparation, and application. Enzym.

Microb. Technol. 1996, 19, 107–117.

(25) Nordlander, J. E.; Njoroge, F. G.; Payne, M. J.; Warman, D. N-(Trifluoroacetyl)--

amino acid chlorides as chiral reagents for Friedel–Crafts synthesis. J. Org. Chem.

1985, 50, 3481–3484.

(26) Di Gioia, M. L.; Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. Facile

approach to enantiomerically pure α-amino ketones by Friedel–Crafts aminoacylation

and their conversion into peptidyl ketones. J. Org. Chem. 2001, 66, 7002–7007.

(27) Balasubramaniam, S.; Aidhen, I. S. The growing synthetic utility of the Weinreb

amide. Synthesis 2008, 3707–3738.

(28) Olah, G. A. Friedel–Crafts and Related Reactions, Volume 1; Interscience Publishers-

John Wiley & Sons, Inc.: London and Beccles, UK, 1963.

(29) Olah, G. A. Friedel–Crafts and Related Reactions, Volume 3 Part 1; Interscience

Publishers-John Wiley & Sons, Inc.: London and Beccles, UK, 1964.

(30) Katritzky, A. R.; Jiang, R.; Suzuki, K. N-Tfa- and N-Fmoc-(-

aminoacyl)benzotriazoles as chiral C-acylating reagents under Friedel–Crafts Reaction

Conditions. J. Org. Chem. 2005, 70, 4993–5000.

(31) Prabhu, G.; Basavaprabhu; Narendra, N.; Vishwanatha, T. M.; Sureshbabu, V. V.

Amino acid chlorides: A journey from instability and racemization toward broader

utility in organic synthesis including peptides and their mimetics. Tetrahedron 2015,

71, 2785–2832.

(32) Nordlander, J. E.; Payne, M. J.; Njoroge, F. G.; Balk, M. A.; Laikos, G. D.;

Vishwanath, V. M. Friedel–Crafts acylation with N-(trifluoroacetyl)--amino acid

chlorides. Application to the preparation of -arylalkylamines and 3-substituted

1,2,3,4-tetrahydroisoquinolines. J. Org. Chem. 1984, 49, 4107–4111.

(33) Wang, L.; Murai, Y.; Yoshida, T.; Okamoto, M.; Tachrim, Z. P.; Hashidoko, Y.;

Hashimoto, M. Utilization of acidic α-amino acids as acyl donors: An effective stereo-

controllable synthesis of aryl-keto α-amino acids and their derivatives. Molecules

2014, 19, 6349–6367.

(34) Tachrim, Z. P.; Wang, L.; Murai, Y.; Yoshida, T.; Kurokawa, N.; Ohashi, F.;

Hashidoko, Y.; Hashimoto, M. Trifluoromethanesulfonic acid as acylation catalyst:

Special feature for C- and/or O-acylation reactions. Catalysts 2017, 7, 40–68.

(35) Effenberger, F.; Epple, G. Catalytic Friedel–Crafts acylation of aromatic compounds.

Angew. Chem. Int. Ed. 1972, 11, 300–301.

(36) Effenberger, F.; Epple, G. Trifluoromethanesulfonic-carboxylic anhydrides, highly

active acylating agents. Angew. Chem. Int. Ed. 1972, 11, 299–300.

(37) Effenberger, F.; Eberhard, J. K.; Maier, A. H. The first unequivocal evidence of the

reacting electrophile in aromatic acylation reactions. J. Am. Chem. Soc. 1996, 118,

12572–12579.

(38) Roberts, R. M. G. Studies in trifluoromethanesulphonic acid–IV: Kinetics and

mechanism of acylation of aromatic compounds. Tetrahedron 1983, 39, 137–142.

(39) Bodanszky, M.; Perlman, D. Peptide antibiotics. Science 1969, 163, 352–358.

159

(40) Bada, J. L.; Schroeder, R. A. Racemization of isoleucine in calcareous marine

sediments: kinetics and mechanism. Earth Planet Sci. Lett. 1972, 15, 1–11.

(41) Dale, J. A.; Mosher, H. S. Nuclear magnetic resonance nonequivalence of

diastereomeric esters of -substituted phenylacetic acids for the determination of

stereochemical purity. J. Am. Chem. Soc. 1968, 90, 3732–3738.

(42) Wishart, D. S.; Sykes, B. D.; Richards, F. M. Improved synthetic methods for the

selective deuteration of aromatic amino acids: Applications of selective protonation

towards the identification of protein folding intermediates through nuclear magnetic

resonance. Biochim. Biophys. Acta 1993, 1164, 36–46.

(43) Atzrodt, J.; Derdau, V.; Fey, T.; Zimmermann, J. The renaissance of H/D exchange.

Angew. Chem. Int. Ed. 2007, 46, 7744–7765.

(44) Beller, M.; Bolm, C. Transition metals for organic synthesis: Building blocks and fine

chemicals; WlLEY-VCH: Weinheim, 1998.

(45) Griffiths, D. V.; Feeney, J.; Roberts, G. C. K.; Burgen, A. S. V. Preparation of

selectively deuterated aromatic amino acids for use in 1H NMR studies of proteins.

Biochim. Biophys. Acta 1976, 446, 479–485.

(46) Munz, D.; Webster-Gardiner, M.; Fu, R.; Strassner, T.; Goddard, W. A.; Gunnoe, T. B.

Proton or metal? The H/D exchange of arenes in acidic solvents. ACS Catal. 2015, 5,

769–775.

(47) Giles, R.; Lee, A.; Jung, E.; Kang, A.; Jung, K. W. Hydrogen–deuterium exchange of

aromatic amines and amides using deuterated trifluoroacetic acid. Tetrahedron Lett.

2015, 56, 747–749.

(48) Giles, R.; Ahn, G.; Jung, K. W. H-D Exchange in deuterated trifluoroacetic acid via

ligand-directed NHC-palladium catalysis: A powerful method for deuteration of

aromatic ketones, amides, and amino acids. Tetrahedron Lett. 2015, 56, 6231–6235.

(49) Murai, Y.; Wang, L.; Masuda, K.; Sakihama, Y.; Hashidoko, Y.; Hatanaka, Y.;

Hashimoto, M. Rapid and controllable hydrogen/deuterium exchange on aromatic

rings of α-amino acids and peptides. Eur. J. Org. Chem. 2013, 5111–5116.

(50) Wang, L.; Murai, Y.; Yoshida, T.; Okamoto, M.; Masuda, K.; Sakihama, Y.;

Hashidoko, Y.; Hatanaka, Y.; Hashimoto, M. Hydrogen/deuterium exchange of cross-

linkable -amino acid derivatives in deuterated triflic acid. Biosci, Biotechnol,

Biochem, 2014, 78, 1129–1134.

(51) Jarosz, S.; Mach, M. Regio- and stereoselective transformations of sucrose at the

terminal positions. Eur. J. Org. Chem. 2002, 769–780.

(52) Khan, R. A., Mufti, K. S. & Parker, K. J. Preparation of sucrose 6,6′-dichloro hexa-

acetate. US4117224A, 1978.

(53) Sachinvala, N. D. 6,6′-Dihalo-6,6′-dideoxy-1′,2,3,3,4,4′-hexa-O-methylsucrose

compounds, US5126438A, 1992.

(54) Waites, G. M. H. A reversible contraceptive action of some 6-chloro-6-deoxy sugars

in the male rat. J. Reprod. Fert. 1978, 52, 153–157.

(55) Raadt, A. De; Sttitz, A. E. A simple convergent synthesis of the mannosidase inhibitor

1 -deoxymannonojirimycin from sucrose. Tetrahedron Lett. 1992, 33, 189–192.

(56) Appel, R. Tertiary phosphane/tetrachloromethane, a versatile reagent for chlorination,

dehydration, and P–N linkage. Angew. Chem. Int. Ed. 1975, 14, 801–811.

(57) Bhattacharjee, M. K.; Mayer, R. M. Interaction of deoxyhalosucrose derivatives with

dextransucrase. Carbohydr. Res. 1985, 142, 277–284.

(58) Chen, C.-C.; Whistler, R. L. Synthesis of 6,6′-dideoxysucrose (6-deoxy-α-d-

glucopyranosyl 6-deoxy-β-d-fructofuranoside). Carbohydr. Res. 1983, 117, 318–321.

(59) Lees, W. J.; Whitesides, G. M. Interpretation of the reduction potential of 6, 6′-

dithiosucrose cyclic disulfide by comparison of the conformations of 6,6′-

dithiosucrose cyclic disulfide, 6,6'-dithiosucrose, and sucrose in aqueous solution. J.

Am. Chem. Soc. 1993, 115, 1860–1869.

160

(60) Hough, L.; Sinchareonkul, L. V.; Richardson, A. C.; Akhtar, F.; Drew, M. G. B.

Bridged derivatives of sucrose: The synthesis of 6,6′-dithiosucrose, 6,6′-

epidithiosucrose and 6,6′-epithiosucrose. Carbohydr. Res. 1988, 174, 145–160.

(61) Luis, J.; Blanco, J.; Manuel, J.; Gadelle, A.; Defaye, J. A mild one-step selective

conversion of primary hydroxyl groups into azides in mono- and oligo-saccharides.

Carbohydr. Res. 1997, 303, 367–372.

(62) Desmaris, L.; Percina, N.; Cottier, L.; Sinou, D. Conversion of alcohols to bromides

using a fluorous phosphine. Tetrahedron Lett. 2003, 44, 7589–7591.

(63) Mach, M. Regio- and stereoselective transformations of sucrose at the terminal. Eur. J.

Org. Chem. 2002, 769–780.

(64) Lichtenthaler, F. W.; Mondel, S. Perspectives in the use of low molecular weight

carbohydrates as organic raw materials. Pure Appl. Chem. 1997, 69, 1853–1866.

(65) Chang, K.; Wu, S.; Wang, K. Regioselective enzymic deacetylation of octa-O-acetyl-

sucrose : Preparation of hepta-O-acetylsucroses. Carbohydr. Res. 1991, 222, 121–129.

(66) Bornemann, S.; Cassells, J. M.; Dordick, J. S.; Hacking, A. J. The use of enzymes to

regioselectively deacylate sucrose esters. Biocatalysis 1992, 7, 1–12.

(67) Tsunekawa, Y.; Masuda, K.; Muto, M.; Muto, Y.; Murai, Y.; Hashidoko, Y.; Orikasa,

Y.; Oda, Y.; Hatanaka, Y.; Hashimoto, M. Chemo-enzymatic synthesis of 1′-

photoreactive sucrose derivates via ether linkage. Heterocycles 2012, 84, 283–290.

(68) Khan, R.; Jenner, M. R.; Lindseth, H. The first replacement of a chlorosulphonyloxy

group by chlorine at C-2 in methyl α-D-glucopyranoside and sucrose derivatives.

Carbohydr. Res. 1980, 78, 173–183.

(69) Hough, L.; Phadnis, S. P.; Tarelli, E. The application of 13

C-N.M.R. Spectroscopy to

products derived from sucrose. Carbohydr. Res. 1976, 47, 151–154.

(70) Ballesteros, A.; Plou, F. J.; Alcalde, M.; Ferrer, M.; Garcıa-Arellano, H.; Reyes-

Duarte, D.; Ghazi, I. Enzymatic synthesis of sugar esters and oligosaccharides from

renewable resources. In biocatalysis in the pharmaceutical and biotechnology

industries, Patel, R. N., Ed.; CRC Press Tylor & Francis Group: Boca Raton, 2007.

(71) Calub, T. M.; Waterhouse, A. L.; Chatterton, N. J. Proton and carbon chemical-shift

assignments for 1-kestose, from two-dimensional N.M.R.-spectral measurements.

Carbohydr. Res. 1990, 199, 11–17.

(72) Siddiqui, I. R.; Purgala, B. Isolation and characterization of oligosaccharides from

honey. Part II. Disaccharides. J. Apic Res. 1967, 7, 51–59.

(73) Hammer, H. The Trisaccharide fraction of some plants belonging to Amaryllidaceae.

Acta. Chem. Scand. 1968, 22, 197–199.

(74) Allen, P. J.; Bacon, J. S. D. Oligosaccharides formed from sucrose by fructose-

transferring enzymes of higher plants. Biochem. J. 1956, 63, 200–206.

(75) Tachrim, Z. P.; Wang, L.; Yoshida, T.; Muto, M.; Nakamura, T.; Masuda, K.;

Hashidoko, Y.; Hashimoto, M. Comprehensive structural analysis of halogenated

sucrose derivatives: Revisiting the reactivity of sucrose primary alcohols.

ChemistrySelect 2016, 1, 58–63.

(76) Pejin, B.; Iodice, C.; Tommonaro, G.; Sabovljevic, M.; Bianco, A.; Tesevic, V.; Vajs,

V.; De Rosa, S. Sugar Composition of the moss Rhodobryum Ontariense (Kindb.)

Kindb. Nat. Prod. Res. 2012, 26, 209–215.

(77) Lichtenthaler, F. W.; Peters, S. Carbohydrates as green raw materials for the chemical

industry. Comptes Rendus Chim. 2004, 7, 65–90.

(78) Liu, F.; Liu, H.; Ke, Y.; Zhang, J. A facile approach to anhydrogalactosucrose

derivatives from chlorinated sucrose. Carbohydr. Res. 2004, 339, 2651–2656.

(79) Fairclough, P. H.; Hough, L.; Richardson, A. C. Derivatives of β-D-fructofuranosyl α-

D-galactopyranoslde. Carbohydr. Res. 1975, 40, 285–298.

(80) Binkley, W. W.; Horton, D.; Bhacca, N. S. Physical studies on oligosaccharides

related to sucrose. Carbohydr. Res. 1969, 155, 245–258.

161

(81) Kakinuma, H.; Yuasa, H.; Hashimoto, H. Glycosyltransfer mechanism of -

glucosyltransferase from Protaminobacter Rubrum. Carbohydr. Res. 1998, 312, 103–

115.

(82) Christofides, J. C.; Davies, D. B.; Martin, J. A.; Rathbone, E. B. Intramolecular

hydrogen bonding in 1′-sucrose derivatives determined by SIMPLE 1H NMR

spectroscopy. J. Am. Chem. Soc. 1986, 108, 5738–5743.

(83) Katritzky, A. R.; Le, K. N. B.; Khelashvili, L.; Mohapatra, P. P. Alkyl, unsaturated,

(hetero)aryl, and N-protected -amino ketones by acylation of organometallic reagents.

J. Org. Chem. 2006, 71, 9861–9864.

(84) Itoh, O.; Honnami, T.; Amano, A.; Murata, K.; Koichi, Y.; Sugita, T. Friedel–Crafts

α-aminoacylation of alkylbenzone with a chiral N-carboxy-α-amino acid anhydride

without loss of chirality. J. Org. Chem. 1992, 57, 7334–7338.

(85) Itoh, O.; Amano, A. Friedel–Crafts -aminoacylation of aromatic compounds with a

chiral N-Carboxy--amino acid anhydride (NCA); Part 2. Synthesis 1999, 423–428.

(86) Katritzky, A. R.; Tao, H.; Jiang, R.; Suzuki, K.; Kirichenko, K. Novel syntheses of

chiral - and -amino acid derivatives utilizing N-protected (aminoacyl)benzotriazoles

from aspartic and glutamic acids. J. Org. Chem. 2007, 72, 407–414.

(87) Katritzky, A. R.; Suzuki, K.; Singh, S. K. Highly diastereoselective peptide chain

extensions of unprotected amino acids with N-(Z--aminoacyl)benzotriazoles.

Synthesis 2004, 2645–2652.

(88) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. The use of esters of N-

hydroxysuccinimide in peptide synthesis. J. Am. Chem. Soc. 1964, 86, 1839–1842.

(89) Joullie, M. M.; Lassen, K. M. Evolution of amide bond formation. Arkivoc 2010, 189–

250.

(90) Wrona-Piotrowicz, A.; Cegliński, D.; Zakrzewski, J. Active esters as acylating

reagents in the Friedel–Crafts reaction: Trifluoromethanesulfonic acid catalyzed

acylation of ferrocene and pyrene. Tetrahedron Lett. 2011, 52, 5270–5272.

(91) Jass, P. A.; Rosso, V. W.; Racha, S.; Soundararajan, N.; Venit, J. J.; Rusowicz, A.;

Swaminathan, S.; Livshitz, J.; Delaney, E. J. Use of N-trifluoroacetyl-protected amino

acid chlorides in peptide coupling reactions with virtually complete preservation of

stereochemistry. Tetrahedron 2003, 59, 9019–9029.

(92) Curphey, T. J. Trifluoracetylation of amino acids and peptides by ethyl trifluoroacetate.

J. Org. Chem. 1979, 44, 2805–2807.

(93) Deblander, J.; Van Aeken, S.; Jacobs, J.; De Kimpe, N.; Tehrani, K. A. A new

synthesis of benzo [f]isoindole-4,9-diones by radical alkylation and bromomethylation

of 1,4-naphthoquinones. Eur. J. Org. Chem. 2009, 4882–4892.

(94) Di Gioia, M. L.; Leggio, A.; Le Pera, A.; Liguori, A.; Perri, F.; Siciliano, C.

Alternative and chemoselective deprotection of the -amino and carboxy functions of

N-Fmoc-amino acid and N-Fmoc-dipeptide methyl esters by modulation of the molar

ratio in the AlCl3/N,N-dimethylaniline reagent system. Eur. J. Org. Chem. 2004,

4437–4441.

(95) Liu, J.; Ikemoto, N.; Petrillo, D.; Armstrong, J. D. Improved syntheses of α-BOC-

aminoketones from α-BOC-amino-Weinreb amides using a pre-deprotonation protocol.

Tetrahedron Lett. 2002, 43, 8223–8226.

(96) Hwang, J. P.; Surya Prakash, G. K.; Olah, G. A. Trifluoromethanesulfonic acid

catalyzed novel Friedel–Crafts acylation of aromatics with methyl benzoate.

Tetrahedron 2000, 56, 7199–7203.

(97) Neuberger, A. Aspects of the metabolism of glycine and of porphyrins. Biochem. J.

1961, 78, 1–10.

(98) Felig, P. The glucose-alanine cycle. Metabolism 1973, 22, 179–207.

(99) Ookawa, A.; Soai, K. Asymmetric synthesis of optically active threo- and erythro-

pyrrolidinylbenzyl alcohol by the highly stereospecific arylation of (S)-proline and the

162

subsequent highly diastereoselective reduction of the -amino ketone J. Chem. Soc.,

Perkin Trans. 1 1987, 1465–1471.

(100) Wedemeyer, W. J.; Welker, E.; Scheraga, H. A. Proline cis-trans isomerization and

protein folding. Biochemistry 2002, 41, 14637–14644.

(101) McClure, D. E.; Arison, B. H.; Jones, J. H.; Baldwin, J. J. Chiral α-amino ketones

from the Friedel–Crafts reaction of protected amino acids. J. Org. Chem. 1981, 46,

2431–2433.

(102) Tachrim, Z. P.; Oida, K.; Ikemoto, H.; Ohashi, F.; Kurokawa, N.; Hayashi, K.;

Shikanai, M.; Sakihama, Y.; Hashidoko, Y.; Hashimoto, M. Synthesis of chiral TFA-

protected α-amino aryl-ketone derivatives with Friedel–Crafts acylation of α-amino

acid N-hydroxysuccinimide ester. Molecules 2017, 22, 1–14.

(103) Weygand, F.; Ropsch, A. N-Trifluoroacetylamino acids. XIV. N-Trifluoroacetylations

of amino acids and peptides with phenyl trifluoroacetate. Chem. Ber. 1959, 92, 2095–

2099.

(104) Fones, W. S.; Lee, M. Hydrolysis of the N-trifluoroacetyl derivative of several D- and

L-amino acids by acylase I. J. Biol. Chem. 1954, 210, 227–238.

(105) Jagt, R. B. C.; Gómez-Biagi, R. F.; Nitz, M. Pattern-based recognition of heparin

contaminants by an array of self-assembling fluorescent receptors. Angew. Chem. Int.

Ed. 2009, 48, 1995–1997.

(106) Chambers, J. J.; Kurrasch-Orbaugh, D. M.; Parker, M. A.; Nichols, D. E.

Enantiospecific synthesis and pharmacological evaluation of a series of super-potent,

conformationally restricted 5-HT2A/2C receptor agonists. J. Med. Chem. 2001, 44,

1003–1010.

(107) Fones, W. S. Some new N-acyl derivatives of alanine and phenylalanine. J. Org. Chem.

1952, 17, 1661–1665.

(108) Reay, A. J.; Williams, T. J.; Fairlamb, I. J. S. Unified mild reaction conditions for C2-

selective Pd-catalysed tryptophan arylation, including tryptophan-containing peptides.

Org. Biomol. Chem. 2015, 13, 8298–8309.

(109) Weygand, F.; Frauendorfer, E. N-(Trifluoroacetyl)amino acids. XXI. Reductive

elimination of the N-trifluoroacetyl and N-trichloroacetyl groups by sodium

borohydride and applications in peptide chemistry. Chem. Ber. 1970, 103, 2437–2449.

(110) Davis, F. A.; Chai, J. α-Amino cyclic dithioketal mediated asymmetric synthesis of

(S)-(-)-α-(N-p-toluenesulfonyl)aminopropiophenone (N-tosyl cathinone). Arkivoc 2008,

190–203.

(111) Sánchez-Ferrer, Á.; Neptuno Rodríguez-López, J.; García-Cánovas, F.; García-

Carmona, F. Tyrosinase: A comprehensive review of its mechanism. Biochim. Biophys.

Acta 1995, 1247, 1–11.

(112) Ito, S. IFPCS presidential lecture: A chemist’s view of melanogenesis. Pigment Cell

Res. 2003, 16, 230–236.

(113) Mason, H. S. The chemistry of melanin III. Mechanism of the oxidation of

dihydroxyphenylalanine by tyrosinase. J. Biol. Chem. 1948, 172, 83–99.

(114) Solano, F. Melanins: Skin pigments and much more—types, structural models,

biological functions, and formation routes. New J. Sci. 2014, 1–28.

(115) Strack, D.; Vogt, T.; Schliemann, W. Recent advances in betalain research.

Phytochemistry 2003, 62, 247–269.

(116) Gandía-Herrero, F.; García-Carmona, F. Biosynthesis of betalains: Yellow and violet

plant pigments. Trends Plant Sci. 2013, 18, 334–343.

(117) Wyler, H.; Chiovini, J. Die Synthese von cyclodopa (leukodopachrom). Helv. Chim.

Acta 1968, 51, 1476–1494.

(118) Nakagawa, S.; Tachrim, Z. P.; Kurokawa, N.; Ohashi, F.; Sakihama, Y.; Suzuki, T.;

Hashidoko, Y.; Hashimoto, M. pH stability and antioxidant power of cyclodopa and

its derivatives. Molecules 2018, 23, 1943–1952.

163

(119) Junk, T.; Catallo, W. J. Hydrogen isotope exchange reactions involving C-H (D, T)

Bonds. Chem. Soc. Rev. 1997, 26, 401–406.

(120) Klein, P. D.; Boutton, T. W.; Hachey, D. L.; Irving, C. S.; Wong, W. W. The use of

stable isotopes in biosynthetic studies. J. Anim. Sci. 1986, 63, 102–110.

(121) Murashige, R.; Hayashi, Y.; Ohmori, S.; Torii, A.; Aizu, Y.; Muto, Y.; Murai, Y.; Oda,

Y.; Hashimoto, M. Comparisons of O-Acylation and Friedel–Crafts acylation of

phenols and acyl chlorides and Fries rearrangement of phenyl esters in

trifluoromethanesulfonic acid: Effective synthesis of optically active homotyrosines.

Tetrahedron 2011, 67, 641–649.

(122) Olah, G. A.; Prakash, G. K. S.; Molnar, Á.; Sommar, J. Superacid chemistry, Second

Ed.; John Wiley & Sons, Inc: Hoboken, New Jersey, USA, 2009.

(123) Bakoss, H. J.; Ranson, R. J.; Roberts, R. M. G.; Sadri, A. R. Studies in

trifluoromethanesulphonic acid-I. Protonation of aromatic derivatives. Tetrahedron

1982, 38, 623–630.